Extra Sugar on Vancomycin: New Analogues for Combating Multidrug

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2018, 61, 286−304

Extra Sugar on Vancomycin: New Analogues for Combating Multidrug-Resistant Staphylococcus aureus and VancomycinResistant Enterococci Dongliang Guan,†,‡,∇ Feifei Chen,†,∇ Lun Xiong,†,§ Feng Tang,†,‡ Faridoon,† Yunguang Qiu,†,‡ Naixia Zhang,†,‡ Likun Gong,†,‡ Jian Li,§ Lefu Lan,*,†,‡,∥ and Wei Huang*,†,‡,⊥ †

CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, ∥State Key Laboratory of Drug Research, and ⊥Center for Bio-drug Development, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China ‡ University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China § Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China S Supporting Information *

ABSTRACT: Lipophilic substitution on vancomycin is an effective strategy for the development of novel vancomycin analogues against drug-resistant bacteria by enhancing bacterial cell wall interactions. However, hydrophobic structures usually lead to long elimination half-life and accumulative toxicity; therefore, hydrophilic fragments were also introduced to the lipo-vancomycin to regulate their pharmacokinetic/pharmacodynamic properties. Here, we synthesized a series of new vancomycin analogues carrying various sugar moieties on the seventh-amino acid phenyl ring and lipophilic substitutions on vancosamine with extensive structure−activity relationship analysis. The optimal analogues indicated 128−1024-fold higher activity against methicillinsusceptible S. aureus, vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-resistant Enterococci (VRE) compared with that of vancomycin. In vivo pharmacokinetics studies demonstrated the effective regulation of extra sugar motifs, which shortened the half-life and addressed concerns of accumulative toxicity of lipo-vancomycin. This work presents an effective strategy for lipo-vancomycin derivative design by introducing extra sugars, which leads to better antibiotic-like properties of enhanced efficacy, optimal pharmacokinetics, and lower toxicity.

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INTRODUCTION Vancomycin, as the first member of glycopeptide antibiotics, was approved for clinical use in the 1950s.1 Its structure was not fully characterized until 1983.2 In antibacterial history, vancomycin opened a new era for fighting drug-resistant Grampositive bacteria, including multidrug-resistant Staphylococcus aureus (MRSA), and was rewarded as “the last resort to fight against untreatable bacterial pathogens” and “ace antibiotics”.3 In recent years, because of the abuse of antimicrobial agents, drug-resistant bacterial infection has become a life-threatening problem in public health. In an estimated survey, drug-resistant bacterial infection may cause 10 million deaths annually worldwide by 2050.4,5 The emergence of vancomycin-resistant bacteria deteriorates the situation. In 1988, vancomycinresistant Enterococci (VRE) was first reported;6 thereafter, vancomycin-intermediate resistant Staphylococcus aureus © 2017 American Chemical Society

(VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) were discovered in 1997 and 2002.7−12 The spread of MRSA and vancomycin-resistant relevant pathogens resulted in serious concerns regarding the outbreak of drug-resistant bacterial infection, and new antibiotics has become an urgent need for combating these pathogens.13 The new-generation glycopeptide antibiotics derived from vancomycin demonstrated improved antibacterial activities against various vancomycin-resistant strains, thereby providing a strategy to tackle the crisis of drug-resistant infections.14−18 Three semisynthetic vancomycin analogues, Telavancin, Dalbavancin, and Oritavancin, were recently launched on the market in 2009 and 2014 for the treatment of MRSA Received: September 13, 2017 Published: December 15, 2017 286

DOI: 10.1021/acs.jmedchem.7b01345 J. Med. Chem. 2018, 61, 286−304

Journal of Medicinal Chemistry

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Figure 1. Structures of vancomycin and its lipo-analogues. Extra sugar moieties or hydrophilic groups are displayed in red color.

Scheme 1. Synthesis of New Vancomycin Derivatives 5−46a

Reagents and conditions: (i) for synthesis of 3a−3n, (a) 2a−n, DIPEA, DMF, room temperature or 55 °C, 2−4 h; (b) NaCNBH3, TFA, MeOH, room temperature, 1 h (for 3a, additional treatment of 20% piperidine in DMF for 20 min); for synthesis of 3o−3p, (c) 2o−p, DIPEA, DMF, 0 °C, 2 h; (ii) HCHO, 4a−k, DIPEA, H2O:MeCN = 1:1, −10 °C, 12 h.

a

infection.19−21 These analogues are also called lipoglycopeptides because their structures bear a linear lipid or biphenyl hydrophobic appendix (Figure 1). These lipo-structures anchor the antibiotics to the bacterial cell wall and dramatically enhance the antibacterial effect.22 The rigid structure of chlorobiphenyl substitution could directly inhibit transglycosylase23 and contribute to the binding with pentaglycyl bridge segments of cell wall peptidoglycan.24 However, hydrophobic groups caused a longer half-life and lower clearance rate in vivo that may lead to accumulative toxicity such as nephrotoxicity.25,26 Hence, hydrophilic groups such as extra sugar moieties (Figure 1) were usually introduced to the scaffold to optimize their pharmacokinetics and toxicity.27 In structures of Dalbavancin, Oritavancin, and Teicoplanin (Figure 1), extra sugar moieties such as mannose, N-

acetylglucosamine, and epi-vancosamine were substituted onto different hydroxyls of the glycopeptide core and play important roles in structural optimization. These extra sugar substitutions were all derived from the corresponding fermentation products28−31 by biosynthesis, which hampers the diversification of various sugar substitutions for new analogue design. Telavancin carries a phosphonomethylaminomethyl group as the hydrophilic tail onto the phenyl of the seventh-amino acid via a selective Mannich reaction.32 With the same reaction, direct assembly of an extra sugar glucosamine26 was previously reported, but the antibacterial activities of these analogues were significantly reduced even with a linear lipid tail on vancosamine. These results implicated the systematic structure−activity relationship (SAR) investigation with various sugar moieties and hydrophobic fragments are required for this 287



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strategy. Recently, the Haldar group reported16,17 a series of novel vancomycin analogues employing “lipophilic-vancomycin-carbohydrate” conjugation by coupling of different sugars onto the C-terminal carboxylic acid and introducing linear lipid groups onto vancosamine. The successful improvement of antibacterial activity against VISA and VRE demonstrated the assembly of extra sugar moieties also modulated the drug efficacy. These examples indicated that introducing the extra sugar to vancomycin by chemical modification presented a new strategy for structural optimization on vancomycin analogues. However, there are still many questions to address, such what kind of sugar to assemble, what position on vancomycin to introduce the sugar, especially when a rigid lipophilic tail like chlorobiphenyl was linked on vancosamine, and how the extra sugar moieties influence the activity and pharmacokinetics. We sought to answer these questions by investigation on precise SAR of extra-sugar-bearing vancomycin analogues.

yl-vancomycin). In vitro anti-MRSA and anti-VISA assays suggested GalN, Gal, and orGlc were the optimal sugar structures; therefore, in the second-round structural modification, we chose these three saccharides to investigate SAR with various lipophilic fragments on vancosamine including linear alkyls and acyls and rigid hydrophobic structures containing benzene rings (compounds 22−46). Some of the lipofragments were selected from previous reports on the basis of the SAR studies.25,31 The full structures of compounds 5−46 are listed in Table 1. To understand how the C-terminus modification influences the antibacterial activity of he lipo-vancomycin-carbohydrate structure, we introduced dimethylaminopropylamine (derived from Dalbavancin) to the C-terminus carboxylic acid by coupling reaction and synthesized compounds 54−56 (Scheme 3 and Table 2). We also utilized aminoglycoside antibiotics (kanamycin and amikacin), which consist of trisaccharide-like motifs, to assemble them onto vancomycin (compounds 58− 63) and test their antibacterial activities (see details in the Supporting Information). NMR Characterization of Vancomycin Analogues. Although chemical modification on vancomycin seventhamino acid resorcinol via Mannich reaction has been previously reported, and NMR chemical shifts of the aryl protons (Ar− Hs) of the modified position were described in the literature,26,32−34 the detailed assignment of these Ar−Hs with clear 2D spectral data was lacking and not yet reported. Herein, we presented the precise characterization with 2D NMR to confirm the assembly position via Mannich reaction (Figure 2). Ha (6.41 ppm) and Hb (6.25 ppm) of intermediate 3m were assigned based on 1H NMR, 1H−1H COSY, 1H−13C HSQC, and 1H−13C HMBC. In the HMBC spectrum (Figure 2, panel B), Hb-Cα but no Ha-Cα correlation was observed that clearly distinguished these two protons. After Mannich reaction, although the chemical shift of Hb (product 46) changed downfield (6.56 ppm), Hb-Cα correlation was still clearly identified, and the disappearance of Ha suggested it was substituted during the reaction. This is the first time report on the precise identification of vancomycin derivatives via selective Mannich reaction with solid spectral data and is helpful for future characterization on compounds of this type. In Vitro Anti-methicillin-susceptible S. aureus (antiMSSA) and Anti-VISA Assay of Vancomycin Analogues and SAR Analysis. With these newly synthesized vancomycin analogues in hand, we sought to investigate the SAR by in vitro antibacterial assays. First, we chose an MSSA strain (Newman)35−37 and a VISA strain (Mu50)37,38 to perform the assay and measure the MIC90 values of our compounds. As shown in Table 3, for decylaminoethyl-vancomycins (compounds 5−14 and Telavancin), extra sugar moieties of GalN, Gal, and orGlc indicated 4−16-fold higher antibacterial activity than other sugars like Glc, GlcN, Man, ManN, orLac, and so forth at a similar MIC90 level of Telavancin in both Newman and Mu50 strains. For chlorobiphenylmethyl-vancomycins (compounds 15−21), GalN, Gal, and orGlc substitution also led to 8−64fold higher activity than that of other sugars and the phosphonomethylaminomethyl group (derived from Telavancin) in the Newman strain and 4−32-fold higher in the Mu50 strain. For lipophilic structures on vancosamine (compounds 22−46), decyl and biphenyl groups exhibited 8−128-fold better efficacy against MSSA and VISA than other linear or rigid hydrophobic tails (18/36/40 vs 11; 32 vs 13; 46 vs 6). For Cterminus-modified compounds (54−56), MIC90 increased to

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RESULTS Synthesis of New Vancomycin Analogues Carrying an Extra Saccharide Motif. To investigate the role of extra sugar moieties on lipo-vancomycin for PK/PD optimization, we introduced various monosaccharides and a disaccharide to the seventh-amino acid phenyl group of lipo-vancomycin via a twostep synthesis (Schemes 1 and 2). First, decylaminoethyl Scheme 2. Synthesis of Saccharide Intermediates 4a−ka

Reagents and conditions: (i) NIS, TfOH, MS4A, dry DCM, 0 °C to room temperature, 12 h; (ii) MeOH, NaOMe, room temperature, 4 h; (iii) H2, Pd−C, MeOH, 2 h; (iv) BF3Et2O (for 51a, 51b) or SnCl4 (for 51c), dry MeCN, 0 °C to room temperature, 12 h; (v) CH3OH, reflux, 4 h; (vi) 2 N HCl, MeOH, room temperature, 2 h. a

(derived from Telavancin) and chlorobiphenylmethyl (derived from Oritavancin) were assembled onto the amino group of vancomycin by reductive amidation. Then, sugar motifs including glucose (Glc), mannose (Man), galactose (Gal), glucosamine (GlcN), mannosamine (ManN), galactosamine (GalN), and open-ring glucose (orGlc) and lactose (orLac) were attached on the seventh-amino acid resorcinol via Mannich reaction (compounds 5−14 from decylaminoethylvancomycin and compounds 15−21 from chlorobiphenylmeth288



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Table 1. Molecular Structures of Vancomycin Analogues 5−46

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Scheme 3. Synthesis of Vancomycin Analogues 54−56a

a

Reagents and conditions: (i) DIPEA, HBTU, DMF, room temperature, 24 h; (ii) HCHO, 4a or 4j, DIPEA, H2O:MeCN = 1:1, −10 °C, 12 h.

subtypes. For vanA and vanM subtypes, C-terminus modification enhanced the anti-VRE activity in 4−8-fold (55 vs 16; 56 vs 19), but for vanB, C-terminus modification decreased 8− 32-fold. The extra sugar motifs also implicated complicated influence against different VRE subtypes. For vanA, the optimal sugar of GalN/Gal (compounds 16/18/40/46) indicated similar MIC90 compared with no extra sugar compounds (17/44) and phosphonomethylaminomethyl compound 21. For vanM, the extra sugar caused a 2−4-fold decrease in activity compared with that of no extra sugar compounds (16/19 vs 17; 40/46 vs 44) and 8−64-fold decrease compared with phosphonomethylaminomethyl compound (16/19 vs 21). However, for vanB, the optimal sugar modification exhibited excellent activities with MIC90 at 0.06 μg/mL (compounds 40 and 46), which was 1024-fold better than vancomycin, 16-fold of Telavancin, and 4-fold of Oritavancin. These data suggested that VRE subtypes may have diverse drug-resistant mechanisms that caused the different responses to different vancomycin analogues via various modification strategies. The systematic SAR studies would help to understand their individual mechanisms and to develop personalized treatments. After all, the optimal compounds (15/40/46/55) bearing extra sugars exhibited dramatically enhanced activities against VREs, especially for the vanB subtype. In Vivo Antibacterial Assay of Vancomycin Analogues. To test the in vivo efficacy, we established two animal models, lethal challenge and abscess formation assays. Compounds 18, 32, 40, 44, and 46 were selected for lethal challenge assay on a mouse model infected by MRSA strain USA300 LAC.40 Each group contains 15 infected mice and was treated with five selected compounds and control compounds of vancomycin and Telavancin via ip injection at a single dose of 7 mg/kg. During 10 days of monitoring, the survival numbers of each group were recorded as shown in Figure 3. The survival rate of the vancomycin group after 10 days was 6.67% (1/15), whereas that of the compound 18 group was 93.3% (14/15), the same as Telavancin. Treatment of compound 46 also led to a survival rate of 86.7% (13/15), and the other three compounds all indicated much better efficacy than that of vancomycin. For the in vivo abscess formation assay, each group contains 12 mice infected by the VISA strain (Mu50). Compounds 18, 46, vancomycin, and Telavancin were administrated to the infected mice via ip injection twice at 7 mg/kg in a 24 h interval. Five days post infection, the mice were sacrificed, and the liver colony-forming units (CFUs) were counted for each mouse as shown in Figure 4. The vancomycin group did not show a significant difference in the liver CFU level compared with that of the control group, whereas compounds 18, 46, and

Table 2. Molecular Structures of Vancomycin Analogues 54−56

1−2 μg/mL compared with corresponding unmodified compound 16 (MIC90, 0.06 μg/mL) and 19 (MIC90, 0.06 μg/mL), which implicated the limited tolerance on C-terminus modification for biphenyl-vancomycin and suggested the assembling position of extra sugar moieties on seventh-amino acid resorcinol would be better than that on the C-terminus for MSSA and VISA. In summary, the extra sugar attachment combined with suitable lipophilic modifications is an effective strategy for developing new vancomycin analogues against MSSA and VISA. The optimal compounds (18/32/36/40/46) bearing GlcN/Gal/orGlc and decyl/biphenyl groups achieved the best activities as 64−128-fold higher than vancomycin, 8− 16-fold higher than Telavancin, and comparable activity with Oritavancin in MSSA and VISA in vitro models. In Vitro Anti-VRE Assay of Vancomycin Analogues and SAR Analysis. Next, we investigated the anti-VRE activities of selected compounds (Table 4). Five strains of three different subtypes of VRE (vanA, vanM, and vanB)39 were used for the examination. On the basis of the results, compounds containing the rigid hydrophobic tails demonstrated higher activities than the linear lipophilic structures in all three VRE subtypes, but extra sugar moieties and Cterminus modification indicated a different influence in three 290



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Figure 2. NMR characterization and signal assignment of vancomycin analogues. (Panel A) 1H NMR spectra of compounds 3m (upper) and 46 (bottom); (Panel B) 1H−13C HMBC spectra of compounds 3m (left) and 46 (right).

Table 3. In Vitro Activities of Vancomycin Derivatives 5−56 against MSSA and VISAa MIC90 (μg/mL)

MIC90 (μg/mL)

MIC90 (μg/mL)

compd

Newman

Mu 50

compd

Newman

Mu 50

compd

Newman

Mu 50

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 0.5 4 1 1 8 4 0.12 0.5 0.25 2 0.06 0.03 0.03 0.06 0.25

2 2 16 4 4 32 8 0.5 1 1 4 0.25 0.12 0.12 0.12 0.5

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

0.5 0.5 0.12 4 0.12 1 1 4 4 1 1 0.06 0.5 32 16 0.03

0.5 2 0.5 8 0.5 2 2 8 8 4 4 0.25 1 64 32 0.25

37 38 39 40 41 42 43 44 45 46 54 55 56 Vanco. Tela. Orita.

0.25 0.5 2 0.015 0.12 2 0.5 0.015 0.12 0.03 1 2 1 2 0.25 0.06

0.5 2 8 0.12 0.5 4 2 0.12 0.5 0.25 1 4 2 8 1 0.06

a

Newman strain, vancomycin-susceptible MSSA, ATCC 5904;35−37 Mu 50 strain, a Healthcare-associated methicillin-resistant S. aureus (HA-MRSA) and VISA strain isolated in Japan.37,38

in vivo pharmacokinetics (PK) properties in normal CD-1 mice compared with trifluoromethylbiphenyl-vancomycin (3m) and control groups of vancomycin and Telavancin (Table 5). Compound 3m indicated a dramatic increase in T1/2 (∼6 h) and AUC (64,194 h ng mL−1) compared with vancomycin (T1/2 0.6 h, AUC 1242 h ng mL−1) that will cause high accumulation in organs, whereas our four compounds containing extra sugars reduced the half-life ∼2-fold and

the Telavancin groups significantly reduced the liver CFU to a similar lower level. These results demonstrated the in vivo efficacy of our new compounds exceeding vancomycin and achieved similar activity as that of the launched drug Telavancin in MRSA and VISA. In Vivo Pharmacokinetic Assay of Vancomycin Analogues. Furthermore, we selected compounds 18, 32, 40, and 46 to investigate how extra sugar moieties regulate the 291



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Table 4. In Vitro Activities of Vancomycin Derivatives against Resistant Enterococcia MIC (μg/mL) vanA

vanM

compd

Efm-HS0649

Efm-HS06188

Efm-HS0847

Efm-HS08257

vanB (EfmHS-vb01)

9 10 11 12 13 15 16 17 18 19 21 32 36 37 40 44 45 46 54 55 56 Vanco. Tela. Orita.

16 64 64 16 2 0.5 4 2 2 4 2 2 8 8 1 1 1 2 0.5 0.5 1 >128 4 0.25

16 64 64 8 4 1 2 1 2 1 4 1 8 8 1 1 0.5 1 1 1 2 >128 8 0.25

8 32 32 8 2 2 16 4 8 16 2 8 32 32 4 4 2 8 1 2 2 >128 4 1

1 8 8 8 0.12 0.12 4 2 4 8 0.12 4 16 16 2 2 1 2 0.12 0.5 1 >128 0.12 0.5

4 16 16 1 2 2 0.12 ⩽0.06 0.25 1 0.25 0.5 0.5 1 ⩽0.06 ⩽0.06 1 ⩽0.06 2 4 8 64 1 0.25

Figure 4. Liver CFU counts of Mu50 (VISA and MRSA)-infected mice after treatment with vancomycin derivatives in an in vivo abscess formation assay. All compounds and positive controls were administrated via ip injection twice at 7 mg/kg. Statistical significance determined by the Mann−Whitney test (two-tailed): *p < 0.05, **p < 0.01, n.s. indicates no significant difference. Each symbol represents the value for an individual mouse. Horizontal bars indicate the observation means, and dashed lines mark the limits of detection.

Cytotoxicity Assay of Vancomycin Analogues. To evaluate the toxicity of our compounds, we performed cytotoxicity assays of compounds 16 and 46 on human renal proximal tubule epithelial cells (HK-2) and a human liver cell line (HL-7702). As shown in Figure 5, compounds 16 and 46 indicated significant lower toxicity in HL-7702 compared with vancomycin and Telavancin at three tested concentrations. On HK-2 cells, these two compounds exhibited a similar effect on cell viability as that of Telavancin and slightly reduced toxicity compared with that of vancomycin. These data demonstrated the advantage of safety of our selected compounds. Interaction of Vancomycin Analogue 46 with Bacterial Peptidoglycan Precursor Peptide. Vancomycin binds to the peptidoglycan precursor peptide Lys-dAla-dAla on the bacterial cell wall, thereby breaking the cell wall biosynthesis and killing the bacteria.34,41−43 To test whether the attached extra sugar is involved in this peptide ligand recognition, we investigated the interaction of compound 46 with the peptidoglycan precursor tripeptide Ac2Lys-dAla-dAla by NMR

a Efm-HS-0649 and Efm-HS-06188: glycopeptide-resistant E. faecium, vanA phenotype, isolated in China;39 Efm-HS-0847 and Efm-HS08257: glycopeptide-resistant E. faecium, vanM phenotype, isolated in China;39 Efm-HS-vb01: glycopeptide-resistant E. faecium, vanB phenotype, isolated in China.

AUC 3−6-fold, implicating that the extra sugar motifs are good fragments for enhancing in vivo clearance and addressing the concerns of accumulative toxicity. Moreover, comparing their PK properties with Telavancin, these compounds exhibited comparable T1/2 and AUC values, which suggested that the extra sugar attachment is a good strategy for improving the druglike features.

Figure 3. Survival chart of USA300 LAC (MRSA)-infected mice after treatment with vancomycin derivatives in an in vivo lethal challenge assay. All compounds and positive controls were administrated with a single dose of 7 mg/kg via ip injection at the first day of infection. 292



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Table 5. Pharmacokinetic Properties of Selected Compoundsa compd 3m 18 32 40 46 Vanco. Tela.

T1/2 (h) 6.04 3.81 3.72 3.45 2.94 0.60 1.13

± ± ± ± ± ± ±

0.30 0.31 0.70 1.26 1.39 0.21 0.13

AUClast (h ng mL−1) 64194 12112 10666 20105 16631 1242 17143

± ± ± ± ± ± ±

6049 2483 2063 1714 4960 335 5611

AUCINF_obs (h ng mL−1) 67452 12238 13450 24345 20047 1271 17304

± ± ± ± ± ± ±

6633 2486 2725 2804 5489 347 5745

CL_obs (mL min−1 kg−1) 1.24 7.0 6.4 3.45 4.42 68.7 5.1

± ± ± ± ± ± ±

0.12 1.38 1.39 0.40 1.42 18 1.5

MRTINF_obs (h) 6.83 4.18 4.79 4.41 4.21 0.79 1.71

± ± ± ± ± ± ±

0.24 0.34 0.24 1.23 2.08 0.26 0.13

VSS_obs (mL/kg) 509 1754 1826 900 1077 3076 520

± ± ± ± ± ± ±

39 380 334 180 449 175 121

a T1/2, half-life; AUClast, area under the concentration−time curve up to the last sampling time; AUCINF_obs, area under the concentration−time curve up to infinity time; CL_obs, clearance; MRTINF_obs, mean retention time up to infinity time; VSS_obs, volume of distribution at steady state.

Figure 5. Cytotoxicity assays of selected compounds. (Panel A) Cell viability test on HL-7702 cells (human liver cells); (Panel B) cell viability test on HK-2 cells (human renal proximal tubule epithelial cells).

Figure 6. Interaction of compound 46 with bacterial peptidoglycan precursor peptide ligand Ac2Lys-dAla-dAla via 1H−13C HSQC NMR measurement. The spectral signals in red represent solo compound 46 and the signals in green represent the mixture of 46 and peptide ligand. The significantly changed signals of 46 after adding the peptide ligand were displayed in the selected spectral regions and assigned with their position information (x1, x2, x3, x5, V1, V5, Gal C2−5/H2−5).

measurement. Constant-time 1H−13C HSQC NMR analysis was performed for solo compound 46 and after adding the

precursor tripeptide. The changes in chemical shifts represented that the corresponding signals were involved in the 293



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interaction. Figure 6 summarizes all the changed 1H−13C correlation signals observed in this assay. The signals of x1, x2, x3, x5, V1, and V5 significantly changed or disappeared, which means these positions on the original vancomycin structure interacted with the target tripeptide ligand. Interestingly, we also observed a signal change of Gal C3/H3 or C5/H5 (these two signals are difficult to distinguish from each other) that suggested the Gal structure of 46 might also contribute to the interaction with the tripeptide. These results explained a possible mechanism of the extra sugar moiety with enhanced interaction with the bacterial cell wall. Then, we used the molecular modeling program Maestro (Schrödinger) to investigate the binding mode of 46 and the tripeptide. Molecular modeling was performed starting with Xray crystal structure of vancomycin cocrystallized with the precursor tripeptide ligand (PDB ID: 1FVM43). Vancomycin was replaced with 46; then, the complex was optimized via energy minimalization to give a rational binding model (Figure S1). In this model, a H-bond interaction of the Gal motif with tripeptide was observed. Combining the modeling result and NMR data, it is very likely the extra sugar moiety participated in the bacterial cell wall binding and therefore enhanced the antibacterial activity. This mechanism is important and helpful for the future designs on novel vancomycin derivatives against drug-resistant bacteria.

vitro anti-VRE assays on three VRE subtypes indicated different responses to C-terminus-modified and extra-sugar-attached lipo-vancomycin derivatives and suggested diverse drugresistant mechanisms of these subtypes. The optimal vancomycin derivatives exhibited excellent antibacterial activity against MSSA, VISA, and VRE with 64−1024-fold enhanced activity compared with that of vancomycin. We evaluated the in vivo activity of the optimal compounds on two infected mouse models for lethal challenge and abscess formation assays. The mice were infected by retro-orbital injection with MRSA or VISA strains following published literature.37,50−52 In our previous work, treatment of high-dose vancomycin (225 mg/kg within 108 h) showed an effective therapeutic effect to Mu50 infection.37 In the current work, because our compounds exceeded vancomycin in in vitro assays, we lowered the dosage to 7 mg/kg for comparison. In the lethal challenge experiment (Figure 3), the survival rates of compounds 18 and 46 are significantly higher than that of vancomycin. The abscess model is to mimic the S. aureus sepsis, a severe and life-threatening disease caused by S. aureus infection. The abscess was gradually developed and could be most obvious between ∼96 and 120 h after infection with S. aureus.53 We euthanized mice for 5 day to detect the accumulation of abscess in kidney, heart, and liver. The abscess in liver is the most sensitive to antibiotic treatment, thereby providing a good model to evaluate the antibacterial efficacy. Compounds 18 and 46 indicated an excellent therapeutic effect to relieve liver abscess, but vancomycin did not show significant activity (Figure 4). Pharmacokinetic data also showed that the extra sugar significantly regulates the half-life and clearance rate of these analogues, which proved our design strategy (Table 5). In the time-course profiles of the blood drug concentration (Figure S3), all the tested compounds maintain >1 μg/mL concentrations in blood after 4 h, which is significantly higher than their MIC values. These optimal PK properties explained their remarkable in vivo efficacy. The NMR binding experiment elucidated that the extra sugar motif of compound 46 is involved in the D-Ala-D-Ala ligand interaction. This result suggested an interesting mechanism of 46 in enhancing bacterial cell wall attachment. One of the vancomycin-resistant mechanisms is the mutation of D-Ala-DAla to D-Ala-D-Lac on the bacterial cell wall that reduced its binding affinity with vancomycin. The Bogar group14,15 reported a series of novel [Ψ[CH2NH]Tpg4] and [Ψ[C( NH)NH]Tpg4] vancomycin analogues with enhanced binding with D-Ala-D-Lac, which demonstrated fantastic antibacterial activities against VREs. These works implicate the enhancement of D-Ala-D-Ala/D-Ala-D-Lac binding is a good strategy to fight against vancomycin-resistant bacteria. The extra sugar in our analogues could interact with the D-Ala-D-Ala ligand that provides a new modification strategy in vancomycin derivative design. Another possible mechanism of these vancomycin analogues with enhanced antibacterial activities may be due to the conformational change of vancomycin after modification. It is reported54 that subtle alteration of vancomycin hydroxyl groups may lead to a significant change in conformation. On the other hand, the detailed structural mechanism still remains to be elucidated in the future. In summary, we report here an effective strategy to introduce extra sugar moieties onto lipo-vancomycin for enhanced drug efficacy, optimal PK properties, and lower toxicity against drugresistant bacteria including MRSA, VISA, and VRE.

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DISCUSSION AND CONCLUSIONS The carbohydrate structure of vancomycin plays important roles in the in vivo activity, PK properties, and drug distribution.44 Although vancomycin aglycon also indicated good in vitro antibacterial activity,45 glycosylation is still indispensable for vancomycin-based drug development. Previous studies reported various strategies to replace the vancosamine/glucose with diverse sugar moieties by enzymatic glycosylation44,46 or chemical synthesis.47,48 Recently, assembly of extra sugars on the vancomycin C-terminus16,17 or seventhamino acid32 presented alternative chemistry in vancomycin analogue design. Lipidation on vancosamine is an efficient approach to prepare lipo-vancomycin derivatives and has been extensively reported.19−24 Introducing lipid structures to other hydroxyl groups of vancomycin by various catalysts broadened the diversity of lipo-vancomycins.49 The advantage of these lipoanalogues comes with dramatically enhanced antibacterial activities against drug-resistant bacteria. Meanwhile, these compounds usually led to long half-lives and brought concerns about accumulative toxicities.25,26 Recently launched glycopeptide antibiotics (Figure 1) feature with lipophilic fragments and extra sugar motifs on the vancomycin core implicates the balance of lipophilic/hydrophilic groups is important in the structural design. Here, we synthesized a vancomycin analogue library with various sugar motifs and lipophilic structures (Schemes 1 and 3) to understand the SAR and optimal combination of lipofragments and extra sugars. The in vitro antibacterial assay revealed that GalN, Gal, and orGlc were the optimal sugar moieties relative to those of other tested sugars for better antibacterial activity, and decyl or biphenyl substitution indicated better activity than that of other lipo structures when combined with the sugar attachment in vancomycin modification. The rigid lipo-vancomycin prefers the assembly of extra sugar moieties on seventh-amino acid resorcinol rather than on the C-terminus for anti-MSSA and VISA activities. In 294



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NaCNBH3 (8 mg, 127 μmol) was dissolved in methanol (1 mL), and the solution was added to the above reaction mixture. TFA (30 μL) was added to adjust the pH to 4. The residue was stirred at room temperature for 2 h to reduce the Schiff base C−N double bond to C− N single bond. In the case of 3a, it required deprotection of Fmoc by treatment with 20% piperidine in DMF for 20 min before performing reductive amination. For intermediates 3o and 3p, vancomycin hydrochloride (100 mg, 67 μmol) and DIPEA (30 μL, 172 μmol) were stirred in DMF (3 mL) until the solution became clear. Then, the mixture was cooled to 0 °C, and R2-COCl 2o−2p (1 equiv) was added in small portions three times every half hour. The residue was stirred at 0 °C for one more hour. Then, the crude product was precipitated by addition of diethyl ether (50 mL) and centrifuged. The supernatant was removed, and the solid cake was washed with diethyl ether (30 mL) once more to give the crude product. The crude was dissolved in water/acetonitrile and was purified by preparative RP-HPLC. The fractions containing target compound were combined and lyophilized to give the product as a white fluffy solid in 20−80% yield. Mannich reaction was performed in the next step to give the final product. R1-NH2 (10 equiv) and DIPEA (800 μmol) were mixed with water (1 mL) and CH3CN (1 mL). The mixture was stirred until the solution was clear. Formaldehyde (37% in water, 4.5 μL, 60 μmol) was added; then, the solution was stirred for 20 min at room temperature. The residue was cooled to −10 °C, and the intermediates 3a−3p (20 μmol) in water (0.5 mL) and CH3CN (0.5 mL) were added to the solution. The reaction was monitored by analytic RP-HPLC. After 12 h, the conversion rate reached 50−80%; then, the residue was treated with TFA to adjust the pH to 2−3. The residue was subject to preparative RP-HPLC purification to give compounds 5−46 as white fluffy solids in 35−95% yield. (α- D-Mannopyranosylethyl)aminomethyl-N-decylaminoethyl Vancomycin (5). Yield 71% (26.5 mg, 14.2 μmol). RT = 16.188 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.56 (br s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.28 (dd, J = 7.2, 0.6 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H),6.84 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.71 (s, 1H), 5.66 (s, 1H), 5.29 (d, J = 7.6 Hz, 1H), 5.26 (s, 1H), 5.12 (m, 3H), 4.83 (s, 1H), 4.65 (s, 2H), 4.44 (s, 1H), 4.40 (s, 1H), 4.11 (br s, 3H), 3.82 (m, 1H), 3.67−3.56 (m, 5H), 3.38 (t, J = 9.4 Hz, 1H), 3.34 (m, 2H), 3.26 (m, 3H), 3.10−3.02 (m, 2H), 2.80 (br s, 2H), 2.54 (s, 3H), 2.18−2.06 (m, 1H), 1.85−1.70 (m, 2H), 1.68−1.58 (m, 2H), 1.54−1.45 (m, 3H), 1.25−1.20 (m, 16H), 1.06 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.2 Hz, 3H), 0.85 (m, 6H). HRMS (ESI) calcd for C87H117Cl2N11O30 [M + 3H]3+ m/z 622.9193, found m/z 622.9264. (β- D-Galactopyranosylethyl)aminomethyl-N-decylaminoethyl Vancomycin (6). Yield 77% (28.7 mg, 15.4 μmol). RT = 16.174 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.57 (br s, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.46 (dd, J = 8.3, 1.8 Hz, 1H), 7.30 (dd, J = 8.1, 3.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.72 (m, 1H), 5.67 (s, 1H), 5.29 (d, J = 7.6 Hz, 1H), 5.25 (s, 1H), 5.14 (s, 1H), 5.11 (d, J = 10.0 Hz, 3H), 4.82 (s, 1H), 4.64 (d, J = 7.0 Hz, 1H), 4.45 (s, 1H), 4.41 (s, 1H), 4.18−4.09 (m, 5H), 3.96 (s, 1H), 3.84−3.75 (m, 1H), 3.67 (d, J = 10.6 Hz, 1H), 3.62 (d, J = 3.0 Hz, 1H), 3.40−3.37 (m, 1H), 3.34−3.23 (m, 5H), 2.83 (br s, 2H), 2.56 (s, 3H), 2.18−2.07 (m, 1H), 1.85−1.70 (m, 2H), 1.67−1.60 (m, 2H), 1.55−1.45 (m, 3H), 1.30 (s, 3H), 1.26−1.20 (m, 16H), 1.07 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (m, 6H). HRMS (ESI) calcd for C87H117Cl2N11O30 [M + 3H]3+ m/z 622.9193, found m/z 622.9115. (β-D-Glucopyranosylethyl)aminomethyl-N-decylaminoethyl Vancomycin (7). Yield 72% (27.1 mg, 14.5 μmol). RT = 16.423 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) 7.78 (m, 1H), 7.59 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.53 (s, 1H), 5.71 (d, J = 7.1 Hz, 1H), 5.67 (s, 1H), 5.30−5.24 (m, 2H), 5.12 (br s, 1H), 5.11− 5.07 (d, J = 12.0 Hz, 2H), 4.81 (s, 1H), 4.68−4.62 (m, 1H), 4.44 (d, J = 5.3 Hz, 1H), 4.40 (d, J = 5.7 Hz, 1H), 4.19 (d, J = 7.8 Hz, 1H), 4.15 (d, J = 7.6 Hz, 1H), 4.12−4.05 (m, 3H), 4.01−3.94 (m, 2H), 3.86−

EXPERIMENTAL SECTION

Material and Instrumentation. All reagents and solvents were commercially purchased from Sinopharm, Bide Pharmatech Ltd., and Shanghai Titan Ltd. (Shanghai, China) and used without further purification. Analytical thin layer chromatography (TLC) was performed on TLC plates precoated with silica gel HSGF254 (200 ± 30 μm thickness). Analytic RP-HPLC analysis was performed on a Beijing Chuang Xin Tong Heng LC-3000 (analytic model) instrument with a C-18 column (5 μm, 4.6 × 150 mm) at 40 °C. The column was eluted with a gradient of 2−90% acetonitrile containing 0.1% TFA in 30 min at a flow rate of 1 mL/min. Preparative RP-HPLC preparation was performed on a Beijing Chuang Xin Tong Heng LC-3000 (preparative model) instrument with a C-18 column (10 μm, 19 × 250 mm) at room temperature. The column was eluted with a gradient of 2−70% acetonitrile containing 0.1% TFA in 30 min at a flow rate of 10 mL/min. HPLC analysis showed that the purities of all final products were more than 95%. 1H NMR, 13C NMR, HSQC, and HMBC spectra were recorded on a BRUKER Ascend 400 or 600 MHz instrument. Chemical shifts were assigned in ppm, and coupling constants were assigned in Hz. Constant-time 1H NMR and HSQC were recorded on an AVANCE III HD BRUKER Ascend 600 MHz instrument. All the final products were added 20 μL of D2O to exchange the active hydrogen of the target products when the solvent was DMSO-d6. ESI-MS spectra were measured with an Agilent 6230 LC-TOF MS spectrometer. Bacterial strains Newman,36 Mu50,38 and USA300 LAC40 were from laboratory stock. Efm-HS-0649, Efm-HS06188, Efm-HS-0847, Efm-HS-08257,39 and VanB(R) were obtained from the Institute of Antibiotics, Huashan Hospital, Fudan University. Female BALB/c mice were purchased from SIPPR-BK Lab Animal Ltd. Male CD-1 mice were purchased from Shanghai Lingchang Biological Technology Co., Ltd. Synthesis and Characterization. Aminoethyl Glycoside (4a− 4d). Compounds 49 and 51a−51c were dissolved in methanol and treated with NaOMe to remove acetyls followed by hydrogenation55 to generate 4a−4d in 70−90% yield. Compounds 4a and 4d were used directly in next step. Aminoethyl β-D-Galactopyranoside 4b. 1H NMR (400 MHz, D2O) δ 4.31 (d, J = 7.8 Hz, 1H), 3.94−3.87 (m, 1H), 3.81 (dd, J = 3.4, 1.0 Hz, 1H), 3.72−3.67 (m, 1H), 3.67−3.64 (m, 1H), 3.60 (ddd, J = 6.9, 5.0, 3.9 Hz, 1H), 3.55 (dd, J = 9.9, 3.5 Hz, 1H), 3.43 (dd, J = 9.9, 7.8 Hz, 1H), 2.94−2.87 (m, 2H). 2-Aminoethyl β-D-Glucopyranoside 4c. 1H NMR (400 MHz, D2O) δ 4.38 (d, J = 7.8 Hz, 1H), 3.90 (ddd, J = 10.6, 5.7, 4.5 Hz, 1H), 3.81 (dd, J = 12.3, 2.3 Hz, 1H), 3.69 (ddd, J = 10.9, 6.1, 4.6 Hz, 1H), 3.61 (dd, J = 12.3, 5.9 Hz, 1H), 3.44−3.33 (m, 2H), 3.29 (d, J = 9.1 Hz, 1H), 3.19 (dd, J = 9.5, 7.9 Hz, 1H), 2.89 (ddd, J = 6.3, 4.4, 1.7 Hz, 2H). Glycolactone Derivatives (4j−4k). D-Gluconic acid lactone (52a, 2 g, 11.2 mmol) or lactobionolactone (52b, 2 g, 5.9 mmol) were refluxed with N-Boc-1,3-propanediamine (1.2 equiv) or N-Cbz-1,3-propanediamine (1.2 equiv) in methanol for 4 h, respectively. The solvent was removed, and the residue was washed with enough ethyl acetate to remove excess N-Boc-1,3-propanediamine or N-Cbz-1,3-propanediamine. The corresponding products were obtained after being dried under a vacuum (see Supporting Information for 1H NMR characterization of these intermediates). Then, the protection groups of Boc and Cbz were removed by 2 N HCl and H2 hydrogenation to give 4j and 4k, respectively, which were used in the next step directly. Gluconic Acid Lactone Derivative (4j). 1H NMR (400 MHz, D2O) δ 4.23 (d, J = 3.4 Hz, 1H), 3.98 (s, 1H), 3.73−3.68 (m, 1H), 3.64 (q, J = 2.0 Hz, 2H), 3.58−3.52 (m, 1H), 3.27 (ddt, J = 26.9, 14.0, 7.1 Hz, 2H), 2.92 (t, J = 7.6 Hz, 2H), 1.85−1.75 (m, 2H). General Procedures for Synthesis of Compounds 5−46. Reductive amination, acylation, and Mannich reactions were used in the synthesis of final compounds successively. First, for intermediates 3a−3n, vancomycin hydrochloride (100 mg, 67 μmol), R2-CHO 2a− 2n (2 equiv), and DIPEA (30 μL, 172 μmol) were stirred in DMF (3 mL) for 2−4 h to form Schiff base at 55 °C. The reaction was monitored by analytic RP-HPLC until complete conversion. Then, 295



Article

Galactosaminylmethyl-N-decylaminoethyl Vancomycin (12). Yield 43% (15.7 mg, 8.6 μmol). RT = 16.685 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.69 (br s, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.59 (br s, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.13 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.71 (d, J = 7.5 Hz, 1H), 5.67 (s, 1H), 5.51 (d, J = 3.6 Hz, 1H), 5.28 (d, J = 4.4 Hz, 1H), 5.26 (d, J = 7.9 Hz, 1H), 5.12 (s, 1H), 5.09 (s, 2H), 4.80 (s, 1H), 4.66 (t, J = 6.8 Hz, 1H), 4.47 (s, 1H), 4.40 (t, J = 5.5 Hz, 1H), 4.23 (s, 1H), 4.00 (s, 1H), 3.84 (dd, J = 10.6, 3.0 Hz, 1H), 3.80 (t, J = 6.5 Hz, 1H), 3.75 (d, J = 3.2 Hz, 1H), 3.65 (d, J = 10.7 Hz, 1H), 3.29−3.21 (m, 4H), 3.20−3.09 (m, 4H), 3.07−3.00 (m, 1H), 2.91 (t, J = 7.7 Hz, 3H), 2.57 (s, 3H), 1.91 (d, J = 11.8 Hz, 1H), 1.82 (d, J = 13.0 Hz, 1H), 1.68−1.59 (m, 2H), 1.56−1.46 (m, 4H), 1.34 (s, 3H), 1.25−1.20 (m, 16H), 1.08 (d, J = 6.2 Hz, 3H), 0.90 (d, J = 5.9 Hz, 4H), 0.85 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C85H113Cl2N11O29 [M + 3H]3+ m/z 608.2439, found m/z 608.2521. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Ndecylaminoethyl Vancomycin (13). Yield 52% (19.7 mg, 10.4 μmol). RT = 16.110 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.62 (s, 1H), 7.79 (s, 1H), 7.60 (br s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 9.6 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.71 (s, 2H), 5.68 (s, 1H), 5.30−5.24 (m, 3H), 5.11 (s, 2H), 5.08 (s, 1H), 4.80 (s, 1H), 4.65 (d, J = 6.0 Hz, 1H), 4.45 (br s, 1H), 4.40 (d, J = 6.0 Hz, 1H), 4.10 (s, 1H), 4.06 (s, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (t, J = 2.9 Hz, 1H), 3.65 (d, J = 10.8 Hz, 1H), 3.57−3.54 (m, 1H), 3.36 (dd, J = 10.8, 5.1 Hz, 1H), 3.28−3.23 (m, 3H), 3.21−3.09 (m, 5H), 2.95−2.88 (m, 4H), 2.56 (s, 3H), 1.86− 1.78 (m, 3H), 1.69−1.59 (m, 2H), 1.56−1.47 (m, 3H), 1.34 (s, 3H), 1.27−1.21 (m, 16H), 1.08 (d, J = 6.1 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.87−0.79 (m, 6H). HRMS (ESI) calcd for C88H120Cl2N12O30 [M + 2H]2+ m/z 948.3883, found m/z 948.3892. (Acyclic-lactobionolactone)acylaminopropylaminomethyl-N-decylaminoethyl Vancomycin (14). Yield 51% (21.0 mg, 10.2 μmol). RT = 16.054 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.62 (s, 1H), 7.79 (s, 1H), 7.61 (s, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.72 (d, J = 7.4 Hz, 1H), 5.68 (s, 1H), 5.29 (s, 1H), 5.27 (d, J = 7.7 Hz, 1H), 5.11 (s, 2H), 5.08 (s, 1H), 4.80 (s, 1H), 4.65 (d, J = 6.7 Hz, 1H), 4.45 (s, 1H), 4.41 (d, J = 5.6 Hz, 1H), 4.27 (d, J = 6.6 Hz, 1H), 4.11 (d, J = 2.3 Hz, 2H), 4.06 (s, 3H), 4.02−3.96 (m, 2H), 3.71−3.62 (m, 3H), 3.61−3.49 (m, 6H), 3.40 (d, J = 6.5 Hz, 1H), 3.32−3.28 (m, 2H), 3.26 (s, 3H), 3.22−3.08 (m, 6H), 3.07−2.97 (m, 1H), 2.94−2.88 (m, 4H), 2.79−2.67 (m, 1H), 2.56 (s, 3H), 1.92 (d, J = 13.4 Hz, 1H), 1.86−1.77 (m, 3H), 1.68−1.61 (m, 2H), 1.55− 1.47 (m, 3H), 1.34 (s, 3H), 1.27−1.21 (m, 16H), 1.09 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.88−0.82 (m, 6H). HRMS (ESI) calcd for C94H130Cl2N12O35 [M + 2H]2+ m/z 1029.4148, found m/z 1029.4148. (β-D-Mannopyranosylethyl)aminomethyl-N-4′-chlorobiphenylmethyl Vancomycin (15). Yield 71% (26.7 mg, 14.2 μmol). RT = 15.744 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.67 (s, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 1.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.3 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.10 (s, 1H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.87 (d, J = 2.1 Hz, 1H), 5.65 (d, J = 7.4 Hz, 1H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.12 (d, J = 12.6 Hz, 2H), 5.07 (d, J = 2.0 Hz, 1H), 4.76 (s, 1H), 4.66 (d, J = 6.7 Hz, 1H), 4.64 (d, J = 1.7 Hz, 1H), 4.46 (s, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.19−4.08 (m, 3H), 4.06−3.99 (m, 1H), 3.98−3.92 (m, 1H), 3.88−3.80 (m, 1H), 3.69−3.60 (m, 3H), 3.57 (t, J = 8.5 Hz, 1H), 3.50 (dd, J = 9.0, 3.4 Hz, 1H), 3.38 (t, J = 9.4 Hz, 1H), 3.36−3.31 (m, 2H), 3.29−3.23 (m, 3H), 3.11 (d, J = 5.6 Hz, 2H), 2.73 (d, J = 15.5 Hz, 1H), 2.10 (d, J = 12.1 Hz, 1H), 1.95−1.86 (m, 1H), 1.82 (d, J = 13.1 Hz, 1H), 1.51 (s, 3H), 1.10 (d, J = 6.2 Hz,

3.77 (m, 1H), 3.70−3.62 (m, 2H), 3.25 (d, J = 5.8 Hz, 3H), 3.14 (dd, J = 10.7, 6.9 Hz, 2H), 3.09 (s, 2H), 3.00 (dt, J = 17.4, 8.9 Hz, 2H), 2.89 (s, 2H), 2.57 (s, 3H), 2.16−2.06 (m, 1H), 1.90 (s, 1H), 1.81 (d, J = 12.5 Hz, 1H), 1.69−1.58 (m, 2H), 1.54−1.48 (m, 3H), 1.32 (s, 3H), 1.25−1.20 (m, 16H), 1.08 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.86−0.80 (m, 6H). HRMS (ESI) calcd for C87H117Cl2N11O30 [M + 3H]3+ m/z 622.9193, found m/z 622.8183. (β-D-Acetylglucosaminylethyl)aminomethyl-N-decylaminoethyl Vancomycin (8). Yield 65% (25.0 mg, 13.1 μmol). RT = 16.201 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.46(d, J = 8.7 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 7.11 (s, 1H), 6.84 (dd, J = 8.5, 1.9 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.51 (s, 1H), 5.66 (s, 1H), 5.61 (s, 1H), 5.25 (d, J = 7.7 Hz, 1H), 5.22 (d, J = 3.8 Hz, 1H), 5.11 (s, 3H), 4.82− 4.74 (m, 1H), 4.68−4.62 (m, 1H), 4.43 (s, 1H), 4.40 (s, 1H), 4.34 (d, J = 8.5 Hz, 1H), 4.16−4.06 (m, 5H), 3.92−3.86 (m, 1H), 3.86−3.79 (m, 1H), 3.71 (d, J = 10.2 Hz, 1H), 3.65 (d, J = 10.6 Hz, 1H), 3.30− 3.21 (m, 5H), 3.18−3.13 (m, 2H), 3.08−2.96(m, 6H), 2.88 (dd, J = 9.4, 6.4 Hz, 2H), 2.76−2.71 (m, 1H), 2.36 (s, 3H), 1.92−1.86 (m, 1H), 1.78 (s, 3H), 1.71 (d, J = 12.9 Hz, 1H), 1.59−1.44 (m, 5H), 1.28 (s, 3H), 1.23−1.19 (m, 16H), 1.04 (d, J = 6.3 Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H), 0.85−0.79 (m, 6H). HRMS (ESI) calcd for C89H120Cl2N12O30 [M + 3H]3+ m/z 636.5948, found m/z 636.5870. Hydroxyethylaminomethyl-N-decylaminoethyl Vancomycin (9). Yield 91% (30.8 mg, 18.1 μmol). RT = 16.450 min (analytical HPLC). 1 H NMR (600 MHz, DMSO-d6) δ 7.81 (d, J = 1.9 Hz, 1H), 7.50 (dd, J = 8.3, 1.8 Hz, 1H), 7.47 (dd, J = 8.3, 1.8 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.12 (s, 1H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.70 (d, J = 7.1 Hz, 1H), 5.64 (s, 1H), 5.26 (d, J = 7.8 Hz, 1H), 5.24 (d, J = 4.0 Hz, 1H), 5.12 (s, 3H), 4.79 (br s, 1H), 4.67−4.63 (m, 1H), 4.45 (d, J = 4.8 Hz, 1H), 4.43 (d, J = 5.8 Hz, 1H), 4.17−4.07 (m, 4H), 3.70−3.64 (m, 3H),3.55 (d, J = 9.0 Hz, 1H), 3.30−3.23 (m, 2H), 3.17 (s, 1H), 3.08 (q, J = 7.3 Hz, 2H), 3.00−2.94 (m, 2H), 2.94−2.89 (m, 2H), 2.13 (q, J = 8.0, 7.6 Hz, 0H), 1.93−1.85 (m, 1H), 1.72 (d, J = 13.1 Hz, 1H), 1.63−1.50 (m, 4H), 1.29 (s, 3H), 1.28−1.19 (m, 16H), 1.08 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.86−0.80 (m, 6H), 1.05 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.87−0.82 (m, 6H). HRMS (ESI) calcd for C81H107Cl2N11O25 [M + 3H]3+ m/z 568.9017, found m/z 568.9059. Mannosaminylmethyl-N-decylaminoethyl Vancomycin (10). Yield 54% (19.7 mg, 10.8 μmol). RT = 16.265 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.54−7.41 (m, 4H), 7.32 (d, J = 8.2 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.13 (s, 1H), 6.86 (d, J = 8.2 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 6.55 (s, 1H), 5.69 (d, J = 7.1 Hz, 1H), 5.64 (s, 1H), 5.26 (d, J = 7.6 Hz, 1H), 5.24 (d, J = 3.9 Hz, 1H), 5.17−5.07 (m, 3H), 4.78 (br s, 1H), 4.69−4.62 (m, 1H), 4.47 (s, 1H), 4.43 (d, J = 5.6 Hz, 1H), 4.28 (s, 1H), 4.12 (d, J = 11.7 Hz, 1H), 3.74−3.62 (m, 3H), 3.57−3.51 (m, 1H), 3.34−3.21 (m, 4H), 3.16 (s, 1H), 3.03 (br s, 1H), 2.93−2.85 (m, 3H), 2.19−2.10 (m, 1H), 1.90 (d, J = 13.1 Hz, 1H), 1.72 (d, J = 13.1 Hz, 1H), 1.59− 1.50 (m, 4H), 1.41−1.34 (m, 1H), 1.29 (s, 3H), 1.27−1.22 (m, 16H), 1.06 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.0 Hz, 3H), 0.87−0.82 (m, 6H). HRMS (ESI) calcd for C85H113Cl2N11O29 [M + 2H]2+ m/z 911.8619, found m/z 911.8621. Glucosaminylmethyl-N-decylaminoethyl Vancomycin (11). Yield 45% (16.6 mg, 9.1 μmol). RT = 16.400 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.79 (d, J = 4.7 Hz, 1H), 7.50−7.41 (m, 2H), 7.31 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (dd, J = 8.5, 2.2 Hz, 1H), 6.76 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.68 (d, J = 7.2 Hz, 1H), 5.63 (s, 1H), 5.48 (d, J = 3.4 Hz, 1H), 5.25 (d, J = 7.7 Hz, 1H), 5.23 (s, 1H), 5.12−5.07 (m, 3H), 4.76 (br s, 1H), 4.67−4.62 (m, 1H), 4.48−4.44 (m, 1H), 4.41 (d, J = 5.5 Hz, 1H), 4.25 (s, 1H), 4.10 (s, 1H), 3.71−3.63 (m, 2H), 3.62−3.57 (m, 2H), 3.30−3.20 (m, 3H), 3.19−3.10 (m, 3H), 2.94−2.84 (m, 4H), 2.18−2.08 (m, 1H), 1.88 (d, J = 10.6 Hz, 1H), 1.71 (d, J = 13.1 Hz, 1H), 1.60−1.50 (m, 4H), 1.44−1.36 (m, 1H), 1.28 (s, 3H), 1.25−1.20 (m, 16H), 1.04 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.2 Hz, 3H), 0.86− 0.78 (m, 6H). HRMS (ESI) calcd for C85H113Cl2N11O29 [M + 3H]3+ m/z 608.2439, found m/z 608.2428. 296



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3H), 0.91 (t, J = 6.6 Hz, 6H). HRMS (ESI) calcd for C88H101Cl3N10O30 [M + 2H]2+ m/z 942.2954, found m/z 942.2943. (β-D-Galactopyranosylethyl)aminomethyl-N-4′-chlorobiphenylmethyl Vancomycin (16). Yield 76% (28.6 mg, 15.2 μmol). RT = 15.840 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.78 (s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.53−7.51 (m, 2H), 7.49 (d, J = 8.6 Hz, 1H), 7.46 (dd, J = 8.3, 1.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.51 (s, 1H), 5.72 (s, 1H), 5.70 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (s, 1H), 5.12 (s, 2H), 5.10 (s, 1H), 4.81 (s, 1H), 4.69−4.62 (m, 1H), 4.45 (s, 1H), 4.40 (s, 1H), 4.21−3.88 (m, 7H), 3.86−3.71 (m, 1H), 3.66 (d, J = 10.6 Hz, 1H), 3.61 (d, J = 3.0 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.53−3.49 (m, 2H), 3.38−3.33 (m, 2H), 3.32−3.22 (m, 5H), 2.52 (s, 3H), 2.17−2.03 (m, 2H), 1.81 (d, J = 13.1 Hz, 1H), 1.69−1.56 (m, 3H), 1.48 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.85 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C88H101Cl3N10O30 [M + 2H]2+ m/z 942.2954, found m/z 942.3032. Hydroxyethylaminomethyl-N-4′-chlorobiphenylmethyl Vancomycin (17). Yield 90% (31.0 mg, 18.0 μmol). RT = 15.920 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.64 (s, 1H), 7.82 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.48−7.45 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.53 (s, 1H), 5.75−5.69 (m, 2H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.12 (s, 2H), 5.09 (d, J = 1.9 Hz, 1H), 4.81 (s, 1H), 4.67−4.63 (m, 1H), 4.46 (d, J = 5.3 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.18−3.96 (m, 6H), 3.68−3.64 (m, 3H), 3.56 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 7.2 Hz, 1H), 3.31−3.21 (m, 4H), 2.98−2.93 (m, 2H), 2.57 (s, 3H), 2.10 (d, J = 9.6 Hz, 2H), 1.81 (d, J = 13.2 Hz, 1H), 1.68−1.66 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.1 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C82H91Cl3N10O25 [M + 3H]3+ m/z 574.5152, found m/z 574.5170. Galactosaminylmethyl-N-4′-chlorobiphenylmethyl Vancomycin (18). Yield 42% (15.4 mg, 8.4 μmol). RT = 15.835 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.80 (br s, 1H), 8.72 (br s, 1H), 7.83 (s, 1H), 7.61 (br s, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.5 Hz, 0H), 7.46 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.13 (s, 1H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 6.53 (s, 1H), 5.72 (d, J = 11.0 Hz, 2H), 5.51 (d, J = 3.6 Hz, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 5.12 (s, 1H), 5.10 (d, J = 2.0 Hz, 2H), 4.80 (s, 1H), 4.66 (q, J = 6.7 Hz, 1H), 4.50−4.44 (m, 1H), 4.40 (t, J = 5.8 Hz, 1H), 4.23 (s, 1H), 4.14 (d, J = 10.3 Hz, 1H), 4.04−3.97 (m, 3H), 3.84 (dd, J = 10.5, 3.0 Hz, 1H), 3.80 (t, J = 6.5 Hz, 1H), 3.75 (d, J = 3.2 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.42−3.38 (m, 1H), 3.30−3.21 (m, 2H), 3.15 (dd, J = 10.5, 3.5 Hz, 1H), 2.76−2.68 (m, 1H), 2.57 (s, 3H), 2.10 (d, J = 13.4 Hz, 1H), 1.82 (d, J = 13.2 Hz, 1H), 1.69−1.60 (m, 2H), 1.49 (s, 3H), 1.22−1.19 (m, 1H), 1.10 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C86H97Cl3N10O29 [M+2H]2+ m/z 920.2822, found m/z 920.2862. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N4′-chlorobiphenylmethyl Vancomycin (19). Yield 55% (21.0 mg, 11.0 μmol). RT = 15.747 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.82 (s, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.49 (dd, J = 8.3, 1.7 Hz, 1H), 7.46 (dd, J = 8.3, 1.7 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (dd, J = 8.4, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.72 (s, 1H), 5.71 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.12 (d, J = 3.8 Hz, 2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.68−4.61 (m, 1H), 4.44 (s, 1H), 4.40 (s, 1H), 4.10 (s, 1H), 4.05 (s, 2H), 4.02 (d, J = 6.7 Hz, 2H), 4.00 (d, J = 3.6 Hz, 2H), 3.89 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.6 Hz, 1H), 3.59−3.53 (m, 3H), 3.36 (dd, J = 10.7, 5.2 Hz, 1H), 3.30−3.23 (m, 3H), 3.22−3.16 (m, 1H), 3.16−3.10 (m, 1H), 2.95−2.87 (m, 2H), 2.55 (s, 3H), 2.15−2.05 (m, 2H), 1.87−1.77 (m, 3H), 1.68−1.59 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H),

0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C89H104Cl3N11O30 [M + 2H]2+ m/z 956.8086, found m/z 956.8070. (Acyclic-lactobionolactone)acylaminopropylaminomethyl-N-4′chlorobiphenylmethyl Vancomycin (20). Yield 51% (21.2 mg, 10.2 μmol). RT = 15.695 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.65 (s, 1H), 7.82 (d, J = 1.9 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 7.62 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.6 Hz, 1H), 7.48− 7.44 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.78−5.68 (m, 2H), 5.34 (d, J = 7.6 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.13 (s, 2H), 5.09 (s, 1H), 4.81 (s, 1H), 4.69−4.62 (m, 1H), 4.44 (d, J = 4.9 Hz, 1H), 4.41 (d, J = 5.7 Hz, 1H), 4.26 (d, J = 6.8 Hz, 1H), 4.10 (d, J = 2.3 Hz, 2H), 4.09−3.97 (m, 8H), 3.71−3.64 (m, 3H), 3.62−3.54 (m, 3H), 3.51 (dd, J = 11.4, 4.7 Hz, 1H), 3.39 (t, J = 6.3 Hz, 1H), 3.33−3.23 (m, 4H), 3.22−3.17 (m, 1H), 3.17−3.11 (m, 1H), 2.92 (t, J = 6.7 Hz, 2H), 2.57 (s, 3H), 2.10 (d, J = 11.1 Hz, 2H), 1.87−1.76 (m, 3H), 1.69−1.60 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C95H114Cl3N11O35 [M + 2H]2+ m/z 1037.8350, found m/z 1037.8290. (Phosphonomethyl)aminomethyl-N-4′-chlorobiphenylmethyl Vancomycin (21). Yield 81% (28.9 mg, 16.3 μmol). RT = 15.855 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.86 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.46 (dd, J = 8.4, 1.9 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.13 (s, 1H), 6.79 (dd, J = 8.4, 2.0 Hz, 1H), 6.72 (d, J = 8.6 Hz, 1H), 6.44 (s, 1H), 5.75 (s, 1H), 5.63 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.28 (s, 1H), 5.14 (d, J = 8.4 Hz, 2H), 5.09 (s, 1H), 4.86 (s, 1H), 4.69−4.62 (m, 1H), 4.43 (s, 1H), 4.41 (s, 1H), 4.31−4.24 (m, 2H), 4.16 (s, 2H), 4.02 (s, 2H), 3.67 (d, J = 10.8 Hz, 1H), 3.56 (d, J = 8.5 Hz, 1H), 3.30−3.21 (m, 3H), 2.70−2.60 (m, 2H), 2.55 (s, 1H), 2.08 (d, J = 22.6 Hz, 2H), 1.82 (d, J = 13.1 Hz, 1H), 1.64 (s, 2H), 1.48 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C81H90Cl3N10O27P [M + 3H]3+ m/z 591.1671, found m/z 591.1677. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Nnonyl Vancomycin (22). Yield 52% (19.1 mg, 10.4 μmol). RT = 14.193 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.81 (s, 1H), 7.61 (br s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.53 (s, 1H), 5.72 (s, 1H), 5.70 (s, 1H), 5.30 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 1.2 Hz, 1H), 5.11 (s, 2H), 5.07 (s, 1H), 4.79 (s, 1H), 4.09 (s, 1H), 4.08− 4.03 (m, 3H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.7, 2.2 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 3.58−3.48 (m, 4H), 3.36 (dd, J = 10.6, 4.9 Hz, 1H), 3.28−3.21 (m, 3H), 3.21−3.17 (m, 1H), 3.17−3.11 (m, 1H), 2.96−2.88 (m, 2H), 2.74 (s, 1H), 2.68 (s, 1H), 2.54 (s, 3H), 2.15− 2.07 (m, 1H), 1.96 (d, J = 11.9 Hz, 1H), 1.86−1.80 (m, 1H), 1.77 (d, J = 13.1 Hz, 1H), 1.68−1.61 (m, 2H), 1.55−1.46 (m, 3H), 1.33 (s, 3H), 1.27−1.21 (m, 14H), 1.06 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C85H113Cl2N11O30 [M + 2H]2+ m/z 919.8594, found m/z 919.8655. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Ndecyl Vancomycin (23). Yield 53% (19.6 mg, 10.6 μmol). RT = 14.283 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.77 (br s, 1H), 8.64 (br s, 1H), 7.80 (d, J = 1.8 Hz, 1H), 7.60 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.10 (s, 1H), 6.84 (dd, J = 8.5, 1.9 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.71 (d, J = 8.5 Hz, 1H), 5.69 (s, 1H), 5.30 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 4.2 Hz, 1H), 5.12 (s, 2H), 5.07 (d, J = 2.0 Hz, 1H), 4.80 (s, 1H), 4.62−4.56 (m, 1H), 4.43 (s, 1H), 4.41 (d, J = 5.9 Hz, 1H), 4.11−4.07 (m, 1H), 4.07−4.03 (m, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.6, 2.2 Hz, 1H), 3.65 (d, J = 10.7 Hz, 1H), 3.56 (t, J = 3.0 Hz, 1H), 3.55−3.53 (m, 1H), 3.53−3.50 (m, 1H), 3.50−3.48 (m, 1H), 3.36 (dd, J = 10.7, 5.0 Hz, 1H), 3.28−3.23 (m, 3H), 3.21−3.17 (m, 1H), 3.16−3.11 (m, 1H), 2.95−2.88 (m, 2H), 2.77−2.72 (m, 1H), 2.70−2.64 (m, 1H), 2.56 (s, 3H), 2.15−2.07 (m, 1H), 1.96 (d, J = 12.0 Hz, 1H), 1.82 (dt, J = 14.0, 297



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7.0 Hz, 1H), 1.77 (d, J = 12.8 Hz, 1H), 1.68−1.60 (m, 2H), 1.55−1.45 (m, 3H), 1.33 (s, 3H), 1.25−1.19 (m, 16H), 1.06 (d, J = 6.2 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.3 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C86H115Cl2N11O30 [M + 2H]2+ m/z 926.8672, found m/z 926.8674. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N4-ethylbenzyl Vancomycin (24). Yield 46% (16.8 mg, 9.2 μmol). RT = 12.123 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 7.82 (s, 1H), 7.61 (br s, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.75− 5.69 (m, 2H), 5.33 (d, J = 7.6 Hz, 1H), 5.28 (d, J = 2.0 Hz, 1H), 5.12 (s, 2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.63 (d, J = 6.6 Hz, 1H), 4.44 (s, 1H), 4.40 (s, 1H), 4.10 (s, 1H), 4.05 (br s, 3H), 4.00 (d, J = 3.6 Hz, 2H), 3.97−3.91 (m, 1H), 3.90−3.87 (m, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.58−3.52 (m, 2H), 3.50 (d, J = 6.0 Hz, 1H), 3.36 (dd, J = 10.5, 4.8 Hz, 1H), 3.30−3.22 (m, 3H), 3.21−3.16 (m, 1H), 3.16−3.11 (m, 1H), 2.92 (s, 2H), 2.61 (q, J = 6.0 Hz, 2H), 2.56 (s, 3H), 2.15−2.05 (m, 2H), 1.85−1.75 (m, 3H), 1.68−1.60 (m, 3H), 1.46 (s, 3H), 1.60 (t, J = 5.4 Hz 3H), 1.09 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C85H105Cl2N11O30 [M + 2H]2+ m/z 915.8281, found m/z 915.8275. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Npentylbenzyl Vancomycin (25). Yield 47% (17.6 mg, 9.4 μmol). RT = 13.959 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.80 (br s, 1H), 8.66 (br s, 1H), 7.84 (s, 1H), 7.62 (br s, 2H), 7.51 (dd, J = 8.4, 1.8 Hz, 1H), 7.48 (dd, J = 8.4, 1.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 2H), 7.32 (d, J = 8.3 Hz, 1H), 7.24 (d, J = 8.4 Hz, 3H), 7.12 (s, 1H), 6.86 (dd, J = 8.5, 1.8 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.78−5.69 (m, 2H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.14 (s, 2H), 5.11 (s, 1H), 4.82 (s, 1H), 4.66 (q, J = 6.6 Hz, 1H), 4.46 (d, J = 3.0 Hz, 1H), 4.43 (s, 1H), 4.42 (s, 1H), 4.14−4.11 (m, 1H), 4.07 (s, 2H), 4.02 (d, J = 3.5 Hz, 1H), 3.96 (t, J = 12.0 Hz, 2H), 3.91 (t, J = 2.9 Hz, 1H), 3.68 (d, J = 10.7 Hz, 1H), 3.60−3.55 (m, 2H), 3.52 (d, J = 7.3 Hz, 1H), 3.38 (dd, J = 12.0, 4.0 Hz, 1H), 3.31−3.24 (m, 2H), 3.24−3.18 (m, 1H), 3.18−3.13 (m, 1H), 2.96−2.90 (m, 2H), 2.59−2.55 (m, 6H), 2.16−2.06 (m, 2H), 1.87−1.78 (m, 3H), 1.70− 1.62 (m, 2H), 1.55 (p, J = 7.5 Hz, 2H), 1.48 (s, 3H), 1.33−1.20 (m, 6H), 1.11 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.1 Hz, 3H), 0.87 (d, J = 6.2 Hz, 3H), 0.85 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C88H111Cl2N11O30 [M + 2H]2+ m/z 936.8515, found m/z 936.8536. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Nbutoxybenzyl Vancomycin (26). Yield 45% (16.9 mg, 9.0 μmol). RT = 12.757 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.66 (br s, 1H), 7.84 (s, 1H), 7.61 (br s, 1H), 7.51 (dd, J = 8.3, 1.8 Hz, 1H), 7.48 (dd, J = 8.3, 1.8 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.77−5.68 (m, 2H), 5.35 (d, J = 7.6 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 5.14 (s, 2H), 5.11 (s, 1H), 4.82 (s, 1H), 4.66 (q, J = 6.6 Hz, 1H), 4.46 (d, J = 5.4 Hz, 1H), 4.42 (s, 1H), 4.15− 4.10 (m, 1H), 4.07 (s, 2H), 4.02 (d, J = 3.6 Hz, 1H), 3.97 (t, J = 6.5 Hz, 2H), 3.94−3.86 (m, 3H), 3.68 (d, J = 10.7 Hz, 1H), 3.60−3.55 (m, 2H), 3.40−3.36 (dd, J = 12.0, 4.3 Hz, 1H), 3.31−3.24 (m, 2H), 3.24−3.18 (m, 1H), 3.18−3.13 (m, 1H), 2.96−2.88 (m, 2H), 2.56 (s, 3H), 2.09 (d, J = 14.8 Hz, 2H), 1.87−1.77 (m, 3H), 1.71−1.64 (m, 5H), 1.47 (s, 3H), 1.42 (h, J = 7.4 Hz, 2H), 1.11 (d, J = 6.3 Hz, 3H), 0.94−0.90 (m, 6H), 0.87 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C87H109Cl2N11O31 [M + 2H]2+ m/z 937.8412, found m/z 937.8439. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N4-(trimethylsilyl)ethynylbenzyl Vancomycin (27). Yield 49% (18.8 mg, 9.9 μmol). RT = 14.595 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.80 (br s, 1H), 8.67 (br s, 1H), 7.84 (s, 1H), 7.63 (br s, 1H), 7.57−7.49 (m, 3H), 7.49−7.42 (m, 3H), 7.32 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.86 (dd, J = 8.4, 1.9 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.55 (s, 1H), 5.77−5.69 (m, 2H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.14 (s, 2H), 5.11 (d, J = 1.9 Hz, 1H), 4.82 (s, 1H), 4.66 (q, J = 6.6 Hz, 1H), 4.46 (d, J = 5.4 Hz, 1H), 4.43 (d, J = 5.7 Hz, 1H), 4.15−4.10 (m, 1H), 4.08 (s,

2H), 4.02 (d, J = 3.6 Hz, 1H), 4.01−3.97 (m, 3H), 3.91 (dd, J = 3.6, 2.3 Hz, 1H), 3.68 (d, J = 10.7 Hz, 1H), 3.60−3.55 (m, 2H), 3.53 (d, J = 7.5 Hz, 1H), 3.39−3.36 (m, 1H), 3.31−3.24 (m, 2H), 3.21 (p, J = 6.8 Hz, 1H), 3.16 (p, J = 6.6 Hz, 1H), 2.94 (t, J = 7.5 Hz, 2H), 2.59 (s, 3H), 2.17−2.07 (m, 2H), 1.89−1.78 (m, 3H), 1.70−1.62 (m, 2H), 1.48 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.93 (d, J = 6.1 Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H), 0.23 (s, 9H). HRMS (ESI) calcd for C88H109Cl2N11O30Si [M + 2H]2+ m/z 949.8322, found m/z 949.8331. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N4-ethynylbenzyl Vancomycin (28). Yield 49% (17.9 mg, 9.8 μmol). RT = 10.384 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.65 (s, 1H), 7.81 (br s, 1H), 7.61 (s, 1H), 7.52 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.2 Hz, 3H), 7.30 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.84 (dd, J = 8.4, 1.9 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.71 (t, J = 6.8 Hz, 2H), 5.32 (d, J = 7.7 Hz, 1H), 5.28 (d, J = 4.1 Hz, 1H), 5.12 (d, J = 3.4 Hz, 2H), 5.08 (d, J = 1.9 Hz, 1H), 4.80 (s, 1H), 4.64 (d, J = 6.7 Hz, 1H), 4.44 (d, J = 5.2 Hz, 1H), 4.41 (d, J = 5.7 Hz, 1H), 4.25− 4.19 (m, 1H), 4.08 (s, 1H), 4.06 (s, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.57−3.53 (m, 2H), 3.50 (d, J = 7.6 Hz, 1H), 3.36 (d, J = 7.0 Hz, 1H), 3.29−3.22 (m, 2H), 3.19 (p, J = 6.7 Hz, 1H), 3.13 (p, J = 13.4, 6.6 Hz, 1H), 2.92 (t, J = 7.4 Hz, 2H), 2.56 (s, 3H), 2.16−2.02 (m, 2H), 1.86−1.75 (m, 3H), 1.68−1.60 (m, 3H), 1.45 (s, 3H), 1.09 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C85H101Cl2N11O30 [M + 2H]2+ m/z 913.8124, found m/z 913.8131. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Ncoumaronemethyl Vancomycin (29). Yield 39% (14.4 mg, 7.8 μmol). RT = 11.220 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.64 (br s, 1H), 7.81 (s, 1H), 7.61 (br s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.46 (dd, J = 8.4, 1.7 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.7 Hz, 2H), 6.52 (s, 1H), 5.72 (s, 1H), 5.71 (s, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 3.9 Hz, 1H), 5.12 (s, 2H), 5.09 (d, J = 2.0 Hz, 1H), 4.80 (s, 1H), 4.66−4.60 (m, 1H), 4.53 (t, J = 8.6 Hz, 2H), 4.45 (d, J = 5.5 Hz, 1H), 4.41 (d, J = 6.2 Hz, 1H), 4.16−4.04 (m, 4H), 4.00 (d, J = 10.5 Hz, 1H), 3.88 (d, J = 12.0 Hz, 2H), 3.69−3.63 (m, 3H), 3.54 (d, J = 8.7 Hz, 1H), 3.29−3.22 (m, 3H), 3.15 (t, J = 8.8 Hz, 2H), 2.98−2.93 (m, 2H), 2.56 (s, 3H), 2.15−2.03 (m, 2H), 1.77 (d, J = 13.2 Hz, 1H), 1.69−1.59 (m, 3H), 1.54−1.48 (m, 1H), 1.45 (s, 3H), 1.22−1.20 (m, 1H), 1.09 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C85H103Cl2N11O31 [M + 2H]2+ m/z 827.2781, found m/z 827.2785. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N2-menaphthyl Vancomycin (30). Yield 48% (17.8 mg, 9.6 μmol). RT = 11.363 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.65 (br s, 1H), 7.99 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.94 (dd, J = 8.0, 7.2 Hz, 1H), 7.90 (dd, J = 8.0, 7.2 Hz, 1H), 7.82 (s, 1H), 7.60 (br s, 1H), 7.58−7.53 (m, 3H), 7.49 (dd, J = 8.3, 1.8 Hz, 1H), 7.46 (dd, J = 8.3, 1.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.9 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.76−5.67 (m, 2H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 5.12 (s, 2H), 5.09 (s, 1H), 4.81 (s, 1H), 4.67 (q, J = 6.6 Hz, 1H), 4.45 (d, J = 5.7 Hz, 1H), 4.41 (d, J = 5.7 Hz, 1H), 4.15 (q, J = 12.8 Hz, 2H), 4.06 (s, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (t, J = 2.9 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.59−3.51 (m, 3H), 3.35 (dd, J = 10.7, 6.0 Hz, 1H), 3.30−3.23 (m, 2H), 3.19 (p, J = 6.6 Hz, 1H), 3.14 (d, J = 6.4 Hz, 1H), 2.92 (t, J = 7.5 Hz, 2H), 2.57 (s, 3H), 2.15−2.08 (m, 2H), 1.86−1.77 (m, 3H), 1.68−1.60 (m, 2H), 1.51 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C87H103Cl2N11O30 [M + 2H]2+ m/z 926.8202, found m/z 926.8226. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N4-pyridylbenzyl Vancomycin (31). Yield 44% (16.5 mg, 8.8 μmol). RT = 12.380 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.72 (d, J = 6.2 Hz, 2H), 8.64 (br s, 1H), 8.22 (d, J = 7.1 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.82 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.62 (br s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 7.10 (s, 1H), 298



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J = 6.0 Hz, 3H). HRMS (ESI) calcd for C86H113Cl2N11O31 [M + 2H]2+ m/z 933.8568, found m/z 933.8577. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Nundecanoyl Vancomycin (35). Yield 45% (16.7 mg, 8.9 μmol). RT = 17.583 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.68 (br s, 1H), 8.55 (br s, 1H), 7.78 (s, 1H), 7.48 (dd, J = 8.2, 1.8 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.85 (dd, J = 8.7, 2.1 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.66 (d, J = 7.8 Hz, 1H), 5.59 (s, 1H), 5.28− 5.19 (m, 3H), 5.16 (s, 1H), 5.12 (s, 2H), 4.75 (br s, 1H), 4.70−4.62 (m, 1H), 4.47 (d, J = 5.8 Hz, 1H), 4.44 (d, J = 6.0 Hz, 1H), 4.14−4.08 (m, 1H), 4.08−4.04 (m, 2H), 4.01 (d, J = 3.5 Hz, 1H), 3.90 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 3.58−3.52 (m, 3H), 3.37 (dd, J = 10.3, 4.5 Hz, 1H), 3.29−3.25 (m, 3H), 3.17−3.11 (m, 2H), 2.97−2.89 (m, 2H), 2.85 (s, 3H), 2.43−2.37 (m, 1H), 2.37−2.30 (m, 1H), 2.15− 2.07 (m, 1H), 1.90 (d, J = 11.4 Hz, 1H), 1.86−1.78 (m, 2H), 1.72 (d, J = 13.1 Hz, 1H), 1.59−1.50 (m, 4H), 1.39 (dq, J = 13.7, 6.9 Hz, 1H), 1.29 (s, 3H), 1.26−1.20 (m, 16H), 1.07 (d, J = 6.4 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.84 (t, J = 6.9 Hz, 3H), 0.81 (d, J = 6.0 Hz, 3H). HRMS (ESI) calcd for C87H115Cl2N11O31 [M + 2H]2+ m/z 940.8647, found m/z 940.8644. Galactosaminylmethyl-N-decyl Vancomycin (36). Yield 40% (14.2 mg, 8.0 μmol). RT = 14.790 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.73 (s, 1H), 7.83 (s, 1H), 7.62 (s, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.15 (s, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.54 (s, 1H), 5.76−5.65 (m, 2H), 5.13 (s, 1H), 5.11 (d, J = 9.2 Hz, 1H), 4.82 (s, 1H), 4.61 (q, J = 6.8 Hz, 1H), 4.52−4.45 (m, 1H), 4.42 (t, J = 5.9 Hz, 1H), 4.25 (s, 1H), 4.16−4.11 (m, 1H), 4.02 (s, 1H), 3.86 (d, J = 10.2 Hz, 1H), 3.82 (t, J = 6.4 Hz, 1H), 3.77 (s, 0H), 3.67 (d, J = 10.8 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.52 (d, J = 9.6 Hz, 1H), 3.30−3.22 (m, 5H), 3.17 (d, J = 10.8 Hz, 1H), 2.79−2.72 (m, 2H), 2.71−2.65 (m, 1H), 2.58 (s, 3H), 1.98 (d, J = 11.9 Hz, 1H), 1.79 (d, J = 13.1 Hz, 1H), 1.71−1.62 (m, 3H), 1.58−1.48 (m, 3H), 1.35 (s, 3H), 1.27−1.20 (m, 16H), 1.08 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.87 (d, J = 6.2 Hz, 3H), 0.86 (t, J = 6.9 Hz, 3H). HRMS (ESI) calcd for C83H108Cl2N10O29 [M + 2H]2+ m/z 890.3408, found m/z 890.3413. Galactosaminylmethyl-N-pentylbenzyl Vancomycin (37). Yield 41% (14.7 mg, 8.2 μmol). RT = 16.038 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.84 (s, 1H), 7.62 (br s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.54 (s, 1H), 5.76−5.68 (m, 2H), 5.52 (d, J = 3.6 Hz, 1H), 5.34 (d, J = 7.6 Hz, 1H), 5.28 (s, 1H), 5.12 (s, 1H), 5.11 (d, J = 5.8 Hz, 1H), 4.81 (s, 1H), 4.65 (q, J = 6.6 Hz, 1H), 4.51−4.45 (m, 1H), 4.41 (t, J = 5.9 Hz, 1H), 4.24 (s, 1H), 4.03−3.98 (m, 1H), 3.94 (q, J = 12.8 Hz, 2H), 3.85 (d, J = 12.5 Hz, 1H), 3.81 (t, J = 6.4 Hz, 1H), 3.76 (d, J = 3.2 Hz, 1H), 3.67 (d, J = 10.7 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.53−3.49 (m, 1H), 3.30−3.22 (m, 2H), 3.16 (d, J = 10.0 Hz, 1H), 2.57 (s, 3H), 2.57−2.52 (m, 2H), 2.10 (d, J = 11.4 Hz, 1H), 1.80 (d, J = 13.2 Hz, 1H), 1.70− 1.61 (m, 2H), 1.54 (p, J = 7.5 Hz, 2H), 1.47 (s, 3H), 1.32−1.19 (m, 5H), 1.10 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C85H104Cl2N10O29 [M + 2H]2+ m/z 900.3252, found m/z 900.3265. Galactosaminylmethyl-N-4-(trimethylsilyl)ethynylbenzyl Vancomycin (38). Yield 41% (15.0 mg, 8.2 μmol). RT = 14.599 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.82 (br s, 1H), 8.72 (br s, 1H), 7.84 (s, 1H), 7.63 (br s, 1H), 7.54−7.49 (m, 3H), 7.49−7.44 (m, 3H), 7.32 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.15 (s, 1H), 6.87 (d, J = 8.9 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.55 (s, 1H), 5.76−5.69 (m, 2H), 5.53 (s, 1H), 5.34 (d, J = 7.6 Hz, 1H), 5.29 (s, 1H), 5.16−5.08 (m, 3H), 4.81 (s, 1H), 4.66 (q, J = 6.8 Hz, 1H), 4.51−4.46 (m, 1H), 4.42 (t, J = 5.9 Hz, 1H), 4.04−3.97 (m, 3H), 3.85 (d, J = 10.7 Hz, 1H), 3.81 (t, J = 6.4 Hz, 1H), 3.76 (s, 1H), 3.68 (d, J = 10.8 Hz, 1H), 3.57 (t, J = 8.5 Hz, 1H), 3.53−3.50 (m, 1H), 3.31−3.23 (m, 2H), 3.17 (d, J = 11.2 Hz, 1H), 2.58 (s, 3H), 2.09 (d, J = 11.7 Hz, 1H), 1.81 (d, J = 13.2 Hz, 1H), 1.69−1.62 (m, 2H), 1.47 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 0.87 (d, J = 6.1

6.87−6.82 (m, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (d, J = 5.9 Hz, 1H), 5.73 (s, 1H), 5.72 (s, 2H), 5.34 (d, J = 7.6 Hz, 1H), 5.29 (s, 1H), 5.12 (s, 2H), 5.11−5.06 (m, 1H), 4.80 (s, 1H), 4.66 (d, J = 7.0 Hz, 1H), 4.49−4.43 (m, 1H), 4.41 (d, J = 5.6 Hz, 1H), 4.13−4.03 (m, 6H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.6, 2.3 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.56 (d, J = 7.0 Hz, 1H), 3.53 (dd, J = 7.0, 2.0 Hz, 0H), 3.36 (dd, J = 10.7, 5.0 Hz, 1H), 3.30−3.22 (m, 3H), 3.21−3.16 (m, 1H), 3.16−3.10 (m, 1H), 2.99 (t, J = 7.9 Hz, 1H), 2.93 (t, J = 7.0 Hz, 1H), 2.85 (t, J = 7.6 Hz, 1H), 2.56 (s, 3H), 2.15−2.06 (m, 2H), 1.97−1.90 (m, 1H), 1.87−1.75 (m, 3H), 1.69−1.61 (m, 2H), 1.49 (s, 3H), 1.21 (s, 1H), 1.11 (d, J = 6.0 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H), 0.85 (d, J = 5.9 Hz, 3H). HRMS (ESI) calcd for C88H104Cl2N12O30 [M + K + H]2+ m/z 959.3037, found m/z 959.3402. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N4′-trifluoromethylbiphenylmethyl Vancomycin (32). Yield 49% (19.1 mg, 9.8 μmol). RT = 16.450 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.83 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.75−5.69 (m, 2H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (s, 1H), 5.12 (s, 2H), 5.10 (s, 1H), 4.80 (s, 1H), 4.69−4.63 (m, 1H), 4.45 (s, 1H), 4.41 (s, 1H), 4.11 (s, 1H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.7, 2.2 Hz, 1H), 3.67 (d, J = 10.6 Hz, 1H), 3.58−3.53 (m, 3H), 3.51 (d, J = 7.0 Hz, 1H), 3.36 (dd, J = 10.3, 4.7 Hz, 1H), 3.30−3.23 (m, 3H), 3.22−3.16 (m, 1H), 3.16− 3.10 (m, 1H), 2.92 (s, 2H), 2.56 (s, 3H), 2.16−2.06 (m, 2H), 1.86− 1.76 (m, 3H), 1.68−1.59 (m, 3H), 1.50 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C90H104Cl2F3N11O30 [M + 2H]2+ m/z 973.8218, found m/z 973.8216. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N3-chloro-4-((2-methyl-[1,1′-biphenyl]-3-yl)methoxyl)benzyl Vancomycin (33). Yield 43% (17.5 mg, 8.6 μmol). RT = 18.750 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.65 (s, 1H), 7.82 (s, 1H), 7.62 (s, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.49 (d, J = 9.1 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 7.38−7.37 (m, 1H), 7.36 (dd, J = 8.7, 1.9 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 7.2, 1.2 Hz, 4H), 7.26 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 9.6 Hz, 1H), 7.19 (d, J = 9.6 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.77−5.64 (m, 2H), 5.33 (d, J = 7.6 Hz, 1H), 5.27 (d, J = 9.4 Hz, 2H), 5.12 (s, 2H), 5.09 (d, J = 2.0 Hz, 1H), 4.80 (s, 1H), 4.68−4.61 (m, 1H), 4.44 (d, J = 5.6 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.13−4.03 (m, 4H), 4.00 (d, J = 3.6 Hz, 2H), 3.93 (s, 2H), 3.89 (dd, J = 3.6, 2.3 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.58−3.53 (m, 2H), 3.51 (d, J = 7.3 Hz, 1H), 3.36 (dd, J = 10.7, 5.0 Hz, 1H), 3.30−3.22 (m, 2H), 3.22−3.17 (m, 1H), 3.16−3.10 (m, 1H), 2.92 (t, J = 7.4 Hz, 2H), 2.78−2.69 (m, 1H), 2.57 (s, 3H), 2.19 (s, 3H), 1.87−1.75 (m, 3H), 1.69−1.60 (m, 2H), 1.54−1.48 (m, 1H), 1.45 (s, 3H), 1.10 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C97H112Cl3N11O31 [M + 2H]2+ m/z 1016.8374, found m/z 1016.8385. (Acyclic-gluconic acid lactone)acylaminopropylaminomethyl-Ndecanoyl Vancomycin (34). Yield 47% (17.5 mg, 9.4 μmol). RT = 16.718 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.69 (br s, 1H), 8.55 (br s, 1H), 7.77 (s, 1H), 7.47 (dd, J = 8.2, 1.8 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.85 (dd, J = 8.7, 2.1 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.66 (d, J = 7.8 Hz, 1H), 5.59 (s, 1H), 5.28− 5.19 (m, 3H), 5.16 (s, 1H), 5.12 (s, 2H), 4.75 (br s, 1H), 4.70−4.62 (m, 1H), 4.47 (d, J = 5.8 Hz, 1H), 4.44 (d, J = 6.0 Hz, 1H), 4.14−4.08 (m, 1H), 4.08−4.04 (m, 2H), 4.01 (d, J = 3.5 Hz, 1H), 3.90 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 3.58−3.52 (m, 3H), 3.37 (dd, J = 10.3, 4.5 Hz, 1H), 3.29−3.25 (m, 3H), 3.23−3.18 (m, 1H) 3.17−3.11 (m, 2H), 2.97−2.89 (m, 2H), 2.85 (s, 3H), 2.43−2.37 (m, 1H), 2.37− 2.30 (m, 1H), 2.15−2.07 (m, 1H), 1.90 (d, J = 11.4 Hz, 1H), 1.86− 1.78 (m, 2H), 1.72 (d, J = 13.1 Hz, 1H), 1.59−1.50 (m, 4H), 1.39 (dq, J = 13.7, 6.9 Hz, 1H), 1.29 (s, 3H), 1.25−1.19 (m, 14H), 1.07 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.83 (t, J = 6.9 Hz, 3H), 0.81 (d, 299



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Hydroxyethylaminomethyl-N-4-pyridylbenzyl Vancomycin (43). Yield 81% (27.3 mg, 16.2 μmol). RT = 12.815 min (analytical HPLC). 1 H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.72 (d, J = 8.0 Hz, 2H), 8.64 (br s, 1H), 8.22 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 1.9 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.61 (br s, 1H), 7.49 (dd, J = 8.4, 1.7 Hz, 1H), 7.46 (dd, J = 8.4, 1.7 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.72 (d, J = 9.5 Hz, 1H), 5.71 (s, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.15−5.11 (m, 2H), 5.10 (s, 1H), 4.81 (s, 1H), 4.66 (q, J = 6.7 Hz, 1H), 4.45 (d, J = 5.3 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.16−4.04 (m, 6H), 4.03−3.96 (m, 1H), 3.69−3.63 (m, 3H), 3.56 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 7.2 Hz, 1H), 3.26 (d, J = 9.6 Hz, 2H), 2.95 (dd, J = 6.4, 4.6 Hz, 2H), 2.13−2.08 (m, 1H), 1.83 (d, J = 13.1 Hz, 1H), 1.68− 1.60 (m, 2H), 1.49 (s, 3H), 1.21 (s, 1H), 1.11 (d, J = 6.1 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C81H91Cl2N11O25 [M + K + H]2+ m/z 863.7630, found m/z 863.8606. Hydroxyethylaminomethyl-N-4′-trifluoromethylbiphenylmethyl Vancomycin (44). Yield 80% (28.1 mg, 16.0 μmol). RT = 16.781 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.80 (br s, 1H), 8.64 (br s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.82 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.62 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 10.1 Hz, 1H), 7.46 (dd, J = 8.4, 1.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.76−5.69 (m, 2H), 5.34 (d, J = 7.6 Hz, 1H), 5.30 (s, 1H), 5.12 (s, 2H), 5.10 (s, 1H), 4.81 (s, 1H), 4.70−4.62 (m, 1H), 4.46 (d, J = 5.2 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.16−3.97 (m, 7H), 3.70−3.63 (m, 3H), 3.56 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 7.1 Hz, 1H), 3.30−3.22 (m, 2H), 2.96 (dd, J = 6.4, 4.7 Hz, 2H), 2.78−2.68 (m, 1H), 2.57 (s, 3H), 2.15−2.07 (m, 2H), 1.82 (d, J = 13.2 Hz, 1H), 1.68−1.59 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C83H91Cl2F3N10O25 [M + 2H]2+ m/z 878.2821, found m/z 878.2834. Hydroxyethylaminomethyl-N-3-chloro-4-((2-methyl-[1,1′-biphenyl]-3-yl)methoxyl)benzyl Vancomycin (45). Yield 78% (28.7 mg, 15.6 μmol). RT = 18.310 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.64 (br s, 1H), 7.82 (s, 1H), 7.62 (s, 1H), 7.57 (d, J = 1.9 Hz, 1H), 7.49 (d, J = 9.1 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 7.39−7.37 (m, 1H), 7.36 (dd, J = 8.7, 1.9 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 7.2, 1.2 Hz, 4H), 7.26 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 9.6 Hz, 1H), 7.19 (d, J = 9.6 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.73 (d, J = 7.3 Hz, 1H), 5.71 (s, 1H), 5.32 (d, J = 7.6 Hz, 1H), 5.29−5.27 (m, 1H), 5.26 (s, 2H), 5.12 (s, 2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.66−4.58 (m, 1H), 4.45 (s, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.16−4.07 (m, 3H), 4.05 (s, 1H), 4.00 (s, 1H), 3.93 (s, 2H), 3.69− 3.62 (m, 3H), 3.55 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 8.8 Hz, 1H), 3.41 (s, 1H), 3.29−3.22 (m, 2H), 2.96 (t, J = 5.6 Hz, 2H), 2.56 (s, 3H), 2.19 (s, 3H), 1.78 (d, J = 13.1 Hz, 1H), 1.64 (d, J = 9.8 Hz, 3H), 1.54− 1.48 (m, 1H), 1.45 (s, 3H), 1.10 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C90H99Cl3N10O26 [M + 2H]2+ m/z 921.2977, found m/z 921.2976. (β-D-Galactopyranosylethyl)aminomethyl-N-4′-trifluoromethylbiphenylmethyl Vancomycin (46). Yield 75% (28.8 mg, 15.0 μmol). RT = 16.518 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.78 (br s, 1H), 8.65 (br s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.83 (s, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 7.8 Hz, 3H), 7.50 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.86 (dd, J = 8.5, 1.8 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.73 (d, J = 7.6 Hz, 1H), 5.71 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.13 (s, 2H), 5.10 (d, J = 2.0 Hz, 2H), 4.82 (s, 1H), 4.67 (q, J = 6.5 Hz, 1H), 4.46 (d, J = 5.4 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.21−4.00 (m, 7H), 3.99−3.93 (m, 1H), 3.83−3.78 (m, 1H), 3.67 (d, J = 10.6 Hz, 1H), 3.62 (d, J = 3.0 Hz, 1H), 3.57 (t, J = 8.5 Hz, 1H), 3.38 (t, J = 6.4 Hz, 1H), 3.33−3.23 (m, 4H), 3.10 (t, J = 5.3 Hz, 2H), 2.58 (s, 3H), 2.12 (d, J = 10.0 Hz, 1H), 1.83 (d, J = 13.1 Hz, 1H), 1.69−1.60 (m, 2H), 1.50 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz,

Hz, 3H), 0.22 (s, 9H). HRMS (ESI) calcd for C85H102Cl2N10O29Si [M + 2H]2+ m/z 913.3058, found m/z 913.3060. Galactosaminylmethyl-N-4-ethynylbenzyl Vancomycin (39). Yield 42% (14.7 mg, 8.4 μmol). RT = 10.494 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.85 (s, 1H), 7.63 (s, 1H), 7.56−7.52 (m, 3H), 7.52−7.45 (m, 3H), 7.33 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 0H), 7.15 (s, 0H), 6.87 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 8.7 Hz, 1H), 6.56 (s, 0H), 5.77−5.68 (m, 2H), 5.54 (d, J = 3.6 Hz, 1H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.3 Hz, 1H), 5.14 (s, 1H), 5.13−5.11 (m, 1H), 4.83 (s, 1H), 4.67 (q, J = 6.6 Hz, 1H), 4.52−4.47 (m, 1H), 4.43 (t, J = 5.9 Hz, 1H), 4.27 (d, J = 1.9 Hz, 2H), 4.06−3.94 (m, 4H), 3.86 (dd, J = 10.6, 3.0 Hz, 1H), 3.82 (t, J = 6.5 Hz, 1H), 3.77 (d, J = 3.3 Hz, 1H), 3.69 (d, J = 10.8 Hz, 1H), 3.58 (t, J = 8.5 Hz, 1H), 3.53 (dd, J = 10.7, 3.6 Hz, 1H), 3.33−3.23 (m, 3H), 3.17 (dd, J = 10.6, 3.4 Hz, 1H), 2.59 (s, 3H), 2.10 (d, J = 11.0 Hz, 1H), 1.82 (d, J = 13.1 Hz, 1H), 1.71−1.61 (m, 3H), 1.48 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C82H94Cl2N10O29 [M + 2H]2+ m/z 877.2860, found m/z 877.2866. Galactosaminylmethyl-N-4′-trifluoromethylbiphenylmethyl Vancomycin (40). Yield 44% (16.5 mg, 8.8 μmol). RT = 16.579 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.83 (br s, 1H), 8.74 (br s, 1H), 7.92 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.5 Hz, 3H), 7.82 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.23 (dd, J = 8.5, 3.3 Hz, 1H), 7.16 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.55 (s, 1H), 5.78−5.69 (m, 2H), 5.53 (d, J = 3.5 Hz, 1H), 5.36 (d, J = 7.7 Hz, 1H), 5.31 (s, 1H), 5.14 (s, 1H), 5.12 (d, J = 4.2 Hz, 1H), 4.83 (s, 1H), 4.68 (q, J = 6.7 Hz, 1H), 4.54−4.46 (m, 1H), 4.43 (t, J = 5.8 Hz, 1H), 4.25 (s, 1H), 4.19−4.12 (m, 1H), 4.08− 4.05 (m, 2H), 4.03 (d, J = 8.6 Hz, 1H), 3.86 (d, J = 11.9 Hz, 1H), 3.82 (t, J = 6.5 Hz, 1H), 3.77 (d, J = 3.1 Hz, 1H), 3.69 (d, J = 10.5 Hz, 1H), 3.58 (t, J = 8.5 Hz, 1H), 3.52 (dd, J = 11.8, 5.6 Hz, 1H), 3.33−3.24 (m, 2H), 3.17 (d, J = 10.6 Hz, 1H), 2.80−2.71 (m, 1H), 2.59 (s, 3H), 2.19−2.07 (m, 2H), 1.84 (d, J = 13.2 Hz, 1H), 1.72−1.60 (m, 2H), 1.51 (s, 3H), 1.13 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C87H97Cl2F3N10O29 [M + 2H]2+ m/z 937.2954, found m/z 937.2962. Hydroxyethylaminomethyl-N-nonyl Vancomycin (41). Yield 80% (26.5 mg, 16.1 μmol). RT = 14.320 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.77 (br s, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.59 (br s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 8.4, 1.8 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.71 (s, 1H), 5.69 (s, 1H), 5.31 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 4.1 Hz, 1H), 5.11 (s, 2H), 5.08 (s, 1H), 4.80 (s, 1H), 4.59 (d, J = 6.6 Hz, 1H), 4.44 (d, J = 5.6 Hz, 1H), 4.40 (s, 1H), 4.17−4.04 (m, 4H), 3.69−3.59 (m, 4H), 3.54 (t, J = 8.5 Hz, 1H), 3.49 (dd, J = 7.0, 2.0 Hz, 1H), 3.25 (s, 3H), 2.94 (br s, 2H), 2.74 (s, 1H), 2.68 (s, 1H), 2.55 (s, 2H), 2.11 (dd, J = 16.4, 9.4 Hz, 1H), 1.97 (d, J = 11.6 Hz, 1H), 1.77 (d, J = 13.2 Hz, 1H), 1.69−1.58 (m, 3H), 1.56−1.44 (m, 4H), 1.33 (s, 3H), 1.25− 1.20 (m, 14H), 1.06 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 4H), 0.85 (d, J = 6.4 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C78H100Cl2N10O25 [M + 2H]2+ m/z 824.3197, found m/z 824.3191. Hydroxyethylaminomethyl-N-4-ethylbenzyl Vancomycin (42). Yield 82% (27.0 mg, 16.4 μmol). RT = 12.408 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.78 (br s, 1H), 7.81 (s, 1H), 7.60 (br s, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.46 (dd, J = 8.3, 1.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 2H), 7.29 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.4, 2.1 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.71 (s, 1H), 5.69 (s, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 4.3 Hz, 1H), 5.12 (s, 2H), 5.09 (d, J = 2.1 Hz, 1H), 4.81 (s, 1H), 4.66−4.60 (m, 1H), 4.45 (d, J = 5.4 Hz, 1H), 4.40 (s, 1H), 4.17−4.03 (m, 4H), 3.92 (q, J = 12.6 Hz, 3H), 3.65 (q, J = 5.2, 4.4 Hz, 3H), 3.54 (d, J = 8.4 Hz, 1H), 3.45 (d, J = 8.7 Hz, 0H), 3.29−3.21 (m, 3H), 2.97−2.91 (m, 2H), 2.59 (q, J = 7.6 Hz, 2H), 2.56 (s, 3H), 2.15−2.02 (m, 2H), 1.79 (d, J = 13.2 Hz, 1H), 1.68−1.61 (m, 2H), 1.46 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.09 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C78H92Cl2N10O25 [M + 2H]2+ m/z 820.2884, found m/z 820.2875. 300



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synthesis of compounds 54−56. Intermediate 3b (50 mg, 30 μmol), HBTU (21 mg, 45 μmol), and DIPEA (25 μL, 151 μmol) were dissolved in DMF (3 mL), and the mixture was stirred for 15 min at rt. Then, N,N-dimethylaminopropylamine (18 μL, 143 μmol) was added, and the solution was stirred for 24 h until analytic RP-HPLC showed the starting material was totally consumed. Then, the crude product was precipitated by addition of diethyl ether (30 mL) and centrifuged. The supernatant was removed, and the solid cake was washed with diethyl ether (15 mL). The crude was dissolved in water and acetonitrile and subjected to preparative RP-HPLC purification. The fractions containing the product were combined and lyophilized to give 54 as a white fluffy solid. Then, the Mannich reaction was performed following the same procedure described above for the synthesis of 5−46 to introduce the Kanamycin and Amikacin fragments. Compounds 58−63 were synthesized directly by the Mannich reaction with compounds 1, 3b, and 3m. All of the products were purified by preparative RP-HPLC to afford the final compounds (55, 56, 58−63) in 30−50% yield. N,N-Dimethyl-N-(3-aminopropyl)-(N-4′-chlorobiphenylmethylvancomycin)carboxamide (54). Yield 30% (15.8 mg, 9.1 μmol). RT = 18.002 min (analytical HPLC). 1H NMR (600 MHz, deuterium oxide) δ 8.71 (s, 1H), 8.54 (s, 1H), 7.84 (d, J = 1.9 Hz, 1H), 7.75− 7.70 (m, 4H), 7.56 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.49− 7.45 (m, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.26 (s, 1H), 7.22 (d, J = 8.3 Hz, 1H), 6.78 (dd, J = 8.4, 1.9 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.39 (d, J = 2.3 Hz, 1H), 6.22 (d, J = 2.3 Hz, 1H), 5.78−5.73 (m, 1H), 5.62 (s, 1H), 5.36 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.5 Hz, 1H), 5.28 (s, 1H), 5.19 (d, J = 3.9 Hz, 1H), 5.18 (s, 1H), 4.93 (s, 1H), 4.71−4.64 (m, 1H), 4.48 (d, J = 5.2 Hz, 1H), 4.32 (d, J = 5.2 Hz, 1H), 4.26 (s, 1H), 4.04 (q, J = 12.7 Hz, 2H), 3.97 (t, J = 7.2 Hz, 1H), 3.68 (d, J = 10.7 Hz, 1H), 3.58 (t, J = 8.5 Hz, 1H), 3.38−3.32 (m, 1H), 3.31−3.23 (m, 2H), 3.15−3.06 (m, 1H), 3.01−2.96 (m, 2H), 2.75 (s, 3H), 2.74 (s, 3H), 2.63 (s, 3H), 2.20−2.09 (m, 2H), 1.88−1.76 (m, 3H), 1.68 (dd, J = 13.2, 6.9 Hz, 1H), 1.65−1.60 (m, 1H), 1.56 (dd, J = 13.2, 6.5 Hz, 1H), 1.51 (s, 3H), 1.13 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.2 Hz, 3H), 0.85 (d, J = 6.3 Hz, 3H). HRMS (ESI) calcd for C84H96Cl3N11O23 [M + 2H]2+ m/z 866.7951, found m/z 866.7988. N,N-Dimethyl-N-(3-aminopropyl)-((α-D-mannopyranosylethyl)aminomethyl-N-4′-chlorobiphenylmethyl-vancomycin)carboxamide (55). Yield 35% (13.8 mg, 7.0 μmol). RT = 16.854 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.67 (s, 1H), 7.83 (s, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.54−7.51 (m, 2H), 7.48−7.45 (m, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.19 (s, 1H), 6.84 (dd, J = 8.7, 1.9 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.49 (s, 1H), 5.73 (d, J = 7.5 Hz, 1H), 5.71 (s, 1H), 5.39 (s, 1H), 5.35 (d, J = 7.6 Hz, 1H), 5.29 (s, 1H), 5.14 (s, 1H), 5.11 (s, 1H), 4.80 (s, 1H), 4.65 (d, J = 7.4 Hz, 1H), 4.64 (d, J = 1.7 Hz, 1H), 4.47 (s, 1H), 4.27 (d, J = 5.1 Hz, 1H), 4.20−3.97 (m, 7H), 3.88−3.80 (m, 1H), 3.69−3.59 (m, 4H), 3.56 (t, J = 8.5 Hz, 1H), 3.50 (dd, J = 9.0, 3.4 Hz, 1H), 3.38 (t, J = 9.4 Hz, 1H), 3.33 (dd, J = 6.5, 3.0 Hz, 1H), 3.29−3.22 (m, 2H), 3.12 (d, J = 19.2 Hz, 3H), 3.04−2.91 (m, 2H), 2.72 (d, J = 1.8 Hz, 6H), 2.57 (s, 3H), 2.15−2.06 (m, 2H), 1.89−1.78 (m, 3H), 1.69−1.59 (m, 2H), 1.50 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C93H113Cl3N12O29 [M + 2H]2+ m/z 984.3479, found m/z 984.3486. N,N-Dimethyl-N-(3-aminopropyl)-((acyclic-gluconic acid lactone)acylaminopropylaminomethyl-N-4′-chlorobiphenylmethylvancomycin)carboxamide (56). Yield 40% (15.9 mg, 8.0 μmol). RT = 16.233 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.67 (s, 1H), 7.82 (m, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.7 Hz, 1H), 7.48−7.44 (m, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.48 (s, 1H), 5.73 (s, 1H), 5.71 (s, 1H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (s, 2H), 5.13 (s, 1H), 5.10 (d, J = 1.9 Hz, 1H), 4.81 (s, 1H), 4.69−4.62 (m, 1H), 4.47 (s, 1H), 4.29 (d, J = 5.4 Hz, 1H), 4.17 (s, 1H), 4.05 (s, 2H), 4.02 (d, J = 11.3 Hz, 2H), 4.00 (d, J = 3.6 Hz, 2H), 3.88 (dd, J = 3.6, 2.3 Hz, 1H), 3.67 (d, J = 10.7 Hz, 1H), 3.59−3.53 (m, 2H), 3.53−3.49 (m, 1H), 3.36 (dd, J = 10.5, 4.9 Hz,

3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C89H101Cl2F3N10O30 [M + 2H]2+ m/z 959.3085, found m/z 959.3090. 2-(Benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-acetyl-α-D mannopyranoside (49). Phenyl-2,3,4,6-tetra-O-acetyl-1-thio-α-D-mannopyranoside (47, 1.1 g, 2.5 mmol) and N-Cbz-ethanolamine (48, 586 mg, 3.0 mmol) were dissolved in anhydrous dichloromethane (20 mL) at 0 °C, and activated 4Å molecular sieves (2 g) were added. The mixture was stirred at 0 °C for 15 min under nitrogen protection. Trifluoromethanesulfonic acid (80 μL, 0.9 mmol) was added, and the solution immediately turned to a red color and stirred at rt overnight. The reaction was quenched with Et3N and diluted with dichloromethane, and the liquid layer was separated by filtration through Celite. The residue was washed with saturated aqueous NaHCO3 and brine successively. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was isolated by column chromatography (EtOAc/ petroleum ether 2:3 then 1:1) to give 49 as a colorless oil in 57% yield. 1 H NMR (400 MHz, CDCl3) δ 7.37−7.27 (m, 5H), 5.34−5.25 (m, 2H), 5.24−5.19 (m, 2H), 5.09 (s, 2H), 4.80 (s, 1H), 4.23 (dd, J = 12.1, 5.8 Hz, 1H), 4.07 (td, J = 12.4, 4.5 Hz, 1H), 3.94 (ddd, J = 8.8, 5.7, 2.4 Hz, 1H), 3.75 (p, J = 4.5 Hz, 1H), 3.55 (ddd, J = 10.5, 6.8, 3.6 Hz, 1H), 3.48−3.32 (m, 2H), 2.13 (s, 3H), 2.06 (s, 4H), 2.01 (d, J = 4.7 Hz, 4H). 2-(Benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-acetyl-β-D-glycoside (51a, 51b). Peracetylated sugar 50a or 50b (1 g, 2.56 mmol) and N-Cbz-ethanolamine 48 (604 mg, 3.09 mmol) were dissolved in dry acetonitrile under a nitrogen atmosphere. The solution was cooled to 0 °C, and BF3·Et2O (1.6 mL, 13.0 mmol) was added dropwise. The reaction was stirred for 30 min at 0 °C and then overnight at rt. The reaction was quenched with Et3N and concentrated in vacuo; the residue was redissolved in dichloromethane and then washed successively with saturated NaHCO3, water, and brine. The organic layer was dried over Na2SO4; the solvent was removed under reduced pressure, and the product was purified by column chromatography on silica. Column chromatography (EtOAc/petroleum ether 2:3) gave pyranoside 51a or 51b as a colorless oil in 30−50% yield. 2-(Benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (51a). 1H NMR (400 MHz, CDCl3) δ 7.39−7.32 (m, 5H), 5.39 (dd, J = 3.4, 1.1 Hz, 1H), 5.21 (t, J = 3.3 Hz, 1H), 5.20−5.16 (m, 1H), 5.11 (s, 2H), 5.05−4.99 (m, 1H), 4.46 (d, J = 7.9 Hz, 1H), 4.18−4.11 (m, 2H), 3.94−3.87 (m, 2H), 3.70 (ddd, J = 10.7, 7.3, 3.7 Hz, 1H), 3.48−3.33 (m, 2H), 2.16 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H). 2-(Benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (51b). 1H NMR (400 MHz, CDCl3) δ 7.36−7.28 (m, 5H), 5.19 (t, J = 9.5 Hz, 1H), 5.09 (s, 2H), 5.06 (t, J = 9.7 Hz, 1H), 4.97 (dd, J = 9.6, 8.0 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H), 4.23 (dd, J = 12.3, 4.9 Hz, 1H), 4.13 (dd, J = 12.0, 2.0 Hz, 1H), 3.86 (ddd, J = 10.0, 5.8, 3.9 Hz, 1H), 3.74−3.63 (m, 2H), 3.45−3.28 (m, 2H), 2.05 (s, 3H), 2.02 (s, 3H), 2.00 (s, 6H). 2-(Benzyloxycarbonyl)aminoethyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside (51c). β-D-Glucosamine pentacetate (50c, 1.0 g, 2.5 mmol) and N-Cbz-ethanolamine (48, 1.25 g, 6.4 mmol) were dissolved in dry acetonitrile (10 mL) under a nitrogen atmosphere. The reaction was cooled to 0 °C, and SnCl4 (360 μL, 3.1 mmol) was added dropwise. The reaction was warmed to rt, heated to 75 °C, and stirred for 14 h. TLC indicated the completion of the reaction; then, the mixture was cooled to rt, quenched with 2 mL of Et3N, and concentrated in vacuum. The residue was dissolved in 50 mL of dichloromethane, washed with water, and with saturated brine successively. The organic phase was then dried over Na2SO4 and concentrated after filtration. Column chromatography (EtOAc/ petroleum ether 1:1 to 4:1) gave a white solid in 30% yield. 1H NMR (400 MHz, CDCl3) δ 7.42−7.30 (m, 5H), 5.57 (d, J = 8.8 Hz, 1H), 5.31 (s, 1H), 5.20 (t, J = 10.0 Hz, 1H), 5.15−5.04 (m, 3H), 4.60 (d, J = 8.3 Hz, 1H), 4.25 (dd, J = 12.3, 4.8 Hz, 1H), 4.15 (dd, J = 12.4, 2.5 Hz, 1H), 3.89 (ddd, J = 10.1, 6.0, 3.4 Hz, 1H), 3.68 (ddd, J = 9.9, 4.8, 2.4 Hz, 2H), 2.08 (s, 3H), 2.05 (d, J = 2.3 Hz, 6H), 1.91 (s, 3H). Kanamycin-Vancomycin and Amikacin-Vancomycin Conjugates (54−56, 58−63). Amidation reactions were performed in the 301



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1H), 3.30−3.23 (m, 2H), 3.22−3.05 (m, 2H), 2.96 (t, J = 7.9 Hz, 2H), 2.90 (d, J = 7.2 Hz, 2H), 2.73 (s, 3H), 2.72 (s, 3H), 2.57 (s, 3H), 2.15−2.07 (m, 2H), 1.83−1.77 (m, 5H), 1.64 (d, J = 10.2 Hz, 2H), 1.50 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C94H116Cl3N13O29 [M + 2H]2+ m/z 998.8612, found m/z 998.8630. Biological Assays. In Vitro Antibacterial Activity for Minimum Inhibitory Concentration (MIC) Determination.56 The MIC values for all antimicrobial agents were measured by broth microdilution using Mueller-Hinton II broth (cation-adjusted, BD 212322). Generally, compounds were dissolved with DMSO to 5.12 mg/mL as stock solutions. All samples were diluted with culture broth to 128 μg/mL as the initial concentration. Further, 1:2 serial dilutions were performed by addition of culture broth to reach concentrations ranging from 128 to 0.0625 μg/mL or lower. Then, 150 μL of each dilution was distributed in 96-well plates as well as sterile controls, growth controls (containing culture broth plus DMSO, without compounds), and positive controls (containing culture broth plus control antibiotics such as vancomycin, Telavancin, etc.). Each test and growth control well was inoculated with 5 μL of an exponential phase bacterial suspension (∼105 CFU/well). The 96-well plates were incubated at 37 °C for 24 h. MIC values of these compounds were defined as the lowest concentration to completely inhibit the bacterial growth. All MIC values were interpreted according to recommendations of the Clinical and Laboratory Standards Institute (CLSI). In Vivo Antibacterial Assay. Mice used in this study were obtained from SIPPR-BK Lab Animal Ltd. (http://www.slarc.org.cn) and housed under specified pathogen-free conditions. Overnight cultured S. aureus USA300 LAC strain (CA-MRSA) or Mu50 strain (HAMRSA/VISA) was transferred into fresh tryptic soy broth (TSB) medium (1:100, v/v) and continued cultivating for 3 h to reach the exponential growth phase. Strains were washed twice with sterile PBS buffer and suspended in the same buffer until use. Six-to-eight week old female BALB/c mice were anesthetized with pentobarbital sodium (80 mg/kg, intraperitoneally) and were infected by retro-orbital injection with a suspension of ∼2.35 × 108 colonyforming units (CFUs) of USA300 LAC (for lethal challenge) or ∼3.8 × 108 CFUs of Mu50 (for abscess formation). For these two animal models, the group used 15 and 12 mice, respectively, as indicated. For the efficacy of selected compounds (18, 32, 40, 44, 46) and control compounds (Vancomycin, Telavancin) to be evaluated on the outcome of S. aureus pathogenesis, mice received intraperitoneal injections with a signal dose of 7 mg/kg of compounds (18, 32, 40, 44, 46, Vancomycin, Telavancin) for USA300 LAC infection or two doses of 7 mg/kg of compounds (18, 46, Vancomycin, Telavancin) dissolved in sterile ddH2O in a 24 h interval for Mu50 infection. The treatment began 1 h after infection. For lethal challenge, the number of dead mice caused by infection was recorded every day and used for the creation of the survival curve. For abscess formation, animals were euthanized by CO2 inhalation 5 d after infection. Liver was harvested and homogenized in 1 mL of sterile PBS buffer (containing 0.01% tritonX-100). Homogenates were serially diluted, and 10 μL dilutions were spotted onto TSA agar to determine the CFU counts. Bacterial burdens within liver were quantified by counting CFU obtained from serial dilutions of the organ homogenate. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Public Health Clinical Center and were performed in accordance with the relevant guidelines and regulations. In Vivo Pharmacokinetic Assay. CD-1 mice used in this study were purchased from Shanghai Lingchang Biological Technology Co. LTD, and the animal room environment was controlled (target conditions: temperature 18−29 °C, relative humidity 30−70%). An electronic time-controlled lighting system was used to provide a 12 h light/12 h dark cycle. Compounds 18, 32, 40, 46, Vancomycin, and Telavancin (5 mg/ kg) were injected in the caudal vein of the tail of fasted CD-1 mice (male, 18−22 g, n = 18, group = 6), respectively. Blood samples were collected at 0.25, 0.75, 2, 4, 8, and 24 h after intravenous administration. Samples were analyzed by Xevo TQ-S triple

quadrupole mass spectrometer (Waters, USA) coupled with ACQUITY I-Class UPLC System (Waters, USA). The ACQUITY UPLC BEH C18 (1.7 μm, 2.0 mm × 50 mm, Waters, USA) was used for the analysis. After analyzing the concentrations of compounds, the values of T1/2, AUClast, AUCINF_obs, CL_obs, MRTINF_obs, and VSS_obs were calculated from time−concentration curves in each animal using Phoenix WinNonlin (CERTARA, USA). Cytotoxicity Assays. Cell viability kit CCK-8 (Cell Counting Kit8)57,58 was used to evaluate the cytotoxicity of newly synthesized vancomycin derivatives. Generally, 100 μL of HK-2 cell (human renal proximal tubule epithelial cells) and HL-7702 cell (human liver cells) suspensions (∼5000 cells/well) were distributed into 96-well plates. After overnight incubation, 10 μL of various concentrations of 16, 46, Vancomycin, and Telavancin were added to the plate well (final concentration: 10, 50, and 100 μM) and further incubated for 72 h. After that, 10 μL of CCK-8 solution was added to each well of the plate, and the whole plate was incubated at 37 °C for 1.5 h. Finally, the optical density at 450 nm (OD450) was recorded using a VERSMax microplate reader. To calculate the cell viability, growth controls (sterile PBS buffer without compounds) and blank controls (culture broth only, no cells) were also included. Each set was performed in triplicate. Cell viability was defined as ODC/ODC=0 × 100, for which ODC represents the optical density of cells treated with different concentrations of compounds and ODC=0 represents the optical density with no compounds added. Interaction of Vancomycin Analogues with Bacterial Peptidoglycan Precursor Peptide. The interaction of compound 46 with bacterial peptidoglycan precursor model peptide was determined by the constant-time [13C,1H]-HSQC. They were recorded on an AVANCE III HD BRUKER Ascend 600 MHz instrument. First, compound 46 (2 mM) dissolved in D2O was determined at 25 °C at pH 3.1; then, the corresponding model peptide ligand Ac2Lys-dAla-dAla was added to make the final concentration up to 10 mM while maintaining the same conditions as above. The solution was determined by the constant-time [13C,1H]-HSQC again.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01345. NMR detection, synthesis, and antibacterial assay of 58− 63, synthetic procedures of intermediates and Telavancin, and NMR, MS, and HPLC spectra of target compounds (PDF) Binding model of 46 and ligand Ac2Lys-dAla-dAla (PDB) Molecular formula strings of all target compounds (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-21-50806600; fax: +86-21-50807088; e-mail: llan@ simm.ac.cn. *Tel: +86-21-20231000 ext. 2517; fax: +86-21-50807088; email: [email protected]. ORCID

Feifei Chen: 0000-0002-7251-1956 Jian Li: 0000-0002-7521-8798 Lefu Lan: 0000-0002-5551-5496 Wei Huang: 0000-0001-6432-1848 Author Contributions ∇

D.G. and F.C. contributed equally to this work.

Notes

The authors declare no competing financial interest. 302



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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NNSFC, Nos. 21372238 and 21572244), the SIMM institute fund (CASIMM0120162014 and CASIMM0120164007), and the Youth Innovation Promotion Association of CAS (No. 2017328). We thank the NMR facility of SIMM for their kind help in compound characterization and NMR binding tests.

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ABBREVIATIONS USED

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REFERENCES

RT, retention time; HBTU, N,N,N′,N′-tetramethyl-O-(1Hbenzotriazol-1-yl)-uronium hexafluorophosphate; MIC, minimum inhibitory concentration; DIPEA, N,N-diisopropylethylamine; MSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus; VISA, vancomycin-intermediate resistant S. aureus; VRSA, vancomycin-resistant S. aureus; VRE, vancomycin-resistant Enterococci; CFU, colony-forming units; 2D, two-dimensional; DMF, dimethylformamide

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